Methods for extending the replicative lifespan of cells

ABSTRACT

The present invention is directed to methods for enhancing the replicative capacity of cells, by culturing the cells in the presence of an active agent or compound which inhibits SIRT1. One method provides expanding stem cells by culturing the cells in the presence of a SIRT1 inhibitor. The resulting cultured cells can be used for a variety of applications including cell-based therapies such as bone marrow transplants, gene therapies, tissue engineering, and in vitro organogenesis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119(e) of U.S.Provisional Patent Application No. 60/690,609 filed Jun. 15, 2005.

FIELD OF THE INVENTION

The present application is directed to methods for extending thereplicative lifespan of cells from a mammalian tissue, preferably forthe ex vivo expansion of stem cells or other cells for cell basedtherapies.

BACKGROUND OF THE INVENTION

Considerable attention has focused on the development of cell-basedtherapies. For example, one type of cell-based therapy includes removingcells or tissue from an individual, manipulating the tissue ex vivo, andreturning the cells to the individual. Treatments include the use oflymphokine activated killer (LAK) cells (see U.S. Pat. No. 4,690,915issued to Rosenberg), tumor infiltrating lymphocytes (TIL) cells (seeU.S. Pat. No. 5,126,132 issued to Rosenberg), cytotoxic T-cells (seeU.S. Pat. No. 6,255,073 issued to Cai, et al.; U.S. Pat. No. 5,846,827issued to Celis, et al.), expanded tumor draining lymph node cells (seeU.S. Pat. No. 6,251,385 issued to Terman), genetically transformed stemcells (see U.S. Pat. No. 6,225,044 issued to Klein, et al.), mononuclearphagocytes (see U.S. Pat. No. 6,210,963 issued to Haddada, et al.),lymphocytes (see U.S. Pat. No. 6,194,207 issued to Bell, et al.; U.S.Pat. No. 5,443,983 issued to Ochoa, et al.; U.S. Pat. No. 6,040,177issued to Riddell, et al.; U.S. Pat. No. 5,766,920 issued to Babbitt, etal.), dendritic cells (see U.S. Pat. No. 6,210,662 issued to Laus, etal.), lymphocytes treated with oxidizing agents (see U.S. Pat. No.6,204,058 issued to Bolton), and cellular vaccines (see U.S. Pat. No.6,227,368 issued to Hiserodt, et al).

The U.S. Food and Drug Administration (FDA) refers to these therapies as“Somatic Cell and Gene Therapies”. As defined by the FDA, a “somaticcell therapy product” can be one or more autologous (self), allogeneic(intra-species), or xenogeneic (inter-species) cell(s) that have beenpropagated, expanded, selected, pharmacologically treated, or otherwisealtered in biological characteristics ex vivo to be administered tohumans and applicable to the prevention, treatment, cure, diagnosis, ormitigation of disease or injuries. A “gene therapy product”, as definedby the FDA, can be one or more products that contain genetic materialwhich are administered to modify and/or manipulate expression of geneticmaterial and/or to alter biological properties of living cells.

The gap between the need for replacement of damaged or diseased organsin patients, with otherwise significant life-expectancy, and the supplyof donor organs is growing at an ever increasing rate (Gridelli andRemuzzi, 2000). Tissue bioengineering and in vitro organogenesisresearch have the potential to bridge this gap. The availability of stemcells for organs in demand would greatly accelerate progress in theseefforts.

A major obstacle to cell-based therapies is the availability ofsufficient numbers of the desired cell type. Even in instances where itis possible to select for relatively purer populations such ashematopoietic stem cells (for example by cell sorting), thesepopulations typically do not expand when cultured.

Accordingly, methods to expand cells ex vivo, particularly withoutsignificant alteration, are highly desirable. The ability to expandpopulations of cells, including a variety of stem cells as well as adultcells such as fibroblasts, beta cells, and cells of the immune system,would greatly contribute to cell-based therapies such as bone marrowtransplants, gene therapies, tissue engineering, and in vitroorganogenesis. Production of autologous stem cells to replace injuredtissue would also reduce the need for immune suppression interventions.Considerable difficulty in achieving this objective has beenencountered, thus far.

Thus, despite the need for methods to expand cells from an individual,including methods to expand them ex vivo, it has not been possible toreadily do so.

SUMMARY OF THE INVENTION

The present invention is directed to methods for enhancing thereplicative capacity of cells, by culturing the cells in the presence ofan active agent or compound which inhibits SIRT1. One embodiment of theinvention provides expanding stem cells or other cells by culturing thecells in the presence of a SIRT1 inhibitor. The resulting cultured cellscan be used for a variety of applications including cell-based therapiessuch as bone marrow transplants, gene therapies, tissue engineering, andin vitro organogenesis.

The present invention provides a method of increasing the replicativecapacity of mammalian cells, comprising culturing the cells in thepresence of an active agent or compound which inhibits SIRT1.

Any compound or agent which inhibits SIRT1 can be used in the methods ofthe invention. In one embodiment, the agent inhibits the activity of theSIRT1. In another embodiment, the agent or compound inhibits SIRT1 bydecreasing transcription. Preferred inhibitors include DNA, RNA, an RNAinterfering agent, PNA, a small organic molecule, a natural product, aprotein, an antibody, a peptide and a peptidomimetic.

In one particularly preferred method, SIRT1 is inhibited by decreasingtranscription by an RNA interfering agent which is a double-stranded,short interfering RNA (siRNA). Preferably, the siRNA is about 15 toabout 28 nucleotides in length. Even more preferably, the siRNA is about19 to about 25 nucleotides in length. The siRNA is about 21 nucleotidesin length. In one embodiment, the siRNA is double-stranded and comprisesa 3′ overhand on each strand. Preferably, the siRNA inhibits SIRT1 bytranscriptional silencing.

The methods of the present invention can be used to enhance thereplicative lifespan of any cells which can divide in culture. Themethods of the present invention can be used to increase the replicativelifespan of any cells for which it is desirable to expand cells invitro, including stem cells and non-stem cells. Preferred cells includeembryonic stem cells, somatic stem cells, umbilical cord blood stemcells, unrestricted somatic stem cells (USSC) derived from humanumbilical cord blood, mesenchymal stem cells, mesenchymal progenitorcells, hematopoietic stem cells, hematopoietic lineage progenitor cells,neural stem cells, neural progenitor cells, endothelial stem cells,endothelial progenitor cells, and fibroblasts. Preferred somatic stemcells include bone marrow derived stem cells, adipose derived stemcells, mesenchymal stem cells, neural stem cells, liver stem cells,hepatocyte precursor cells, pancreatic stem cells, skin stem cells, andcorneal epithelium stem cells.

In one preferred embodiment, the cells are human cells. In anotherpreferred embodiment, the cells are murine cells.

The methods of the present invention enhance the replicative lifespan ofthe cells cultured in the presence of the SIRT1 inhibitor, resulting intheir extended expansion in vitro. Preferably, the cultured cellsundergo at least one mitotic cell division. Even more preferably, thecultured cells undergo at least ten mitotic cell divisions.

In one preferred embodiment, the cultured cells are capable ofself-renewal and expansion in culture, and have the potential todifferentiate into cells of other phenotypes.

One embodiment of the present invention provides cells obtained byculturing cells in the presence of a SIRT1 inhibitor.

The present invention also provides methods of treating a patient inneed of a cell-based therapy, by selecting a patient in need of acell-based therapy; removing cells from said patient or donor; culturingthe cells under conditions which inhibit SIRT1; harvesting the culturedcells; and transplanting the cultured cells into the patient.

One embodiment of the invention provides a method of gene therapy, by a)selecting a patient in need of gene therapy; removing cells from thepatient or donor; culturing the cells under conditions which inhibitSIRT1; transducing DNA into said cultured cells; harvesting saidcultured cells; and transplanting said cultured cells into said patient.

Yet another embodiment of the invention provides methods for screeningfor a compound or agent useful for increasing the replicative lifespanof cells, by screening a library of candidate compounds to identifythose compounds which inhibit SIRT1.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1F show that S1KO MESS are resistant to culture-inducedreplicative senescence, and show impaired passage-induced accumulationof p19^(ARF). FIG. 1A shows numbers of population doublings (PD) overserial passaging according to a 3T3 protocol, carried out in triplicatefor two independent WT and S1KO cell lines. The data is representativeof 6 independent experiments, and for MEFs derived from multiple mouselitters, and from two independent gene-targeted disruptions of SIRT1function (Cheng et al., 2003). FIG. 1B shows colony formation uponseeding at low density (10³ cells/10 cm plate). Quantitation of theaverage number of colonies from three independent experiments isindicated. In all panels, error bars indicate the standard error of themean. FIGS. 1C and 1D show acute inactivation of SIRT1 in conditionallytargeted MEFs by Adenoviral Cre recombinase extends replicativelifespan. FIG. 1C shows Southern and Western analysis of Cre-deletion inex4^(Flox)/S1KO MEFs, following treatment with Adenoviral GFP-Cre orcontrol GFP. For Southern analysis, genomic DNA genomic DNA was digestedwith Bgl II and probed with an internal Eco RI-Sal I fragment (Cheng etal., 2003). Bands corresponding to non-deleted (ex4^(Flox)) and deleted(Δex4) conditional-targeted allele are indicated. In this Southern, theS1KO null allele results in a 3.4 kb band. For Western analysis, thewild-type (wt) and mutant (Δex4) SIRT1 proteins are indicated. FIG. 1Dshows 3T3 serial passaging assays, in triplicate, of ex4^(Flox)/S1KO andex4^(Flox)/WT lines following GFP-Cre deletion or control GFP treatment.FIGS. 1E and F show reintroduction of exogenous SIRT1 into late passageS1KO MEF cultures. FIG. 1E shows Western analysis of SIRT1 proteinlevels following reconstitution with SIRT1 (S1) or control (Co) virus,compared to endogenous levels (WT). FIG. 1F shows serial passagingaccording to 3T3 protocol as in FIG. 1. Arrow indicates retroviraltransduction with SIRT1 or empty virus control at passage 7.

FIGS. 2A-2D show Western analysis. In FIG. 2A, Western analysis ofp19^(ARF), p53, and acetylated p53 levels is shown in passage 3 MEFs, atbase-line or following treatment with 0.2 ug/ml Adriamycin for 8 hours.FIG. 2B shows Western analysis of p19^(ARF), p53 and acetylated p53levels in S1KO and WT control MEFs at passages 2, 5, and 8. FIG. 2Cshows reversal of p19^(ARF) levels upon reconstitution with exogenousSIRT1 at passage 7. FIG. 2D shows Western analysis of p19^(ARF) levelsin SIRT1-, p53-doubly deficient MEFs. In all panels, error bars indicatethe standard error of the mean.

FIGS. 3A-3E show SIRT1 regulates proliferative capacity under chronic,mild oxidative stress. FIG. 3A shows cell-cycle arrest (S-phase ratio,calculated as ratio of BrdU incorporation in treated cells compared tountreated cells) was measured 18 hours after exposure of cultures to theindicated doses of H₂O₂. Data represent the mean of three independentcell lines. FIG. 3B shows proliferation of cells cultured in sub-lethalconcentrations of H₂O₂ (50 uM), or under low serum (3%). FIGS. 3C and Dshow senescence-associated beta-galactosidase activity of cells treatedas in FIG. 3B. FIG. 3E shows levels of p19^(ARF) in cells treated as inFIG. 3B. Co, control; LS, low serum; HP, hydrogen peroxide. In allpanels, error bars indicate the standard error of the mean.

FIGS. 4A-4C show SIRT1 is dispensable for oncogene-induced prematuresenescence. FIG. 4A shows cell-cycle arrest following exposure tooncogenic Ras. S-phase ratio indicates the ratio of S-phase cells inH-Ras^(V12)-treated cells compared to control-treated cells. Datarepresent average of three independent experiments. WT3T3 indicatesimmortalized, late passage WT lines. FIG. 4B shows representative softagar assays of passage 6 (P6) and passage 50 (P50) passage S1KO and WTMEF lines after infection with H-Ras^(V12). Experiments were carried outin triplicate. Average number of colonies following Ras treatment wereas follows: two independent early passage WT lines: 0.3±0.6; threeindependent S1KO lines: 0.3±0.6; late passage 3T3 lines derived from WTMEFs: 21.7±10, 24±7, 23±5; late passage 3T3 lines derived from KO MEFs:0.3±0.6, 0.3±0.6, 0; NIH3T3 cells: 34.3±6.7. FIG. 4C shows Westernanalysis of p19^(ARF) and p21 protein induction following infection withretroviral H-Ras^(V12) (+) or control virus (−) in WT3T3 and S1KO3T3lines, derived from serial passaging according to 3T3 protocols. In allpanels, error bars indicate the standard error of the mean.

FIG. 5 shows that SIRT1 promotes senescence in response to prolongedreplication, but not oncogene activation or acute DNA damage. Acute DNAdamage insults activate p53 by p19^(ARF)-independent mechanisms, whereasreplicative senescence, which is due to chronic, sub-lethal oxidativestress, and Ras-induced premature senescence activate p53 by inducingp19^(ARF). These findings suggest that SIRT1 is required for p19^(ARF)induction only during replicative senescence, in response to chronic,sub-lethal genotoxic stress.

FIGS. 6A-6F show SIRT1-deficiency extends replicative lifespan. FIGS.6A-B show 3T3 serial passaging assays of conditional SIRT1 MEFs as inFIG. 2. FIG. 6A shows ex4^(Flox)/S1KO and ex4^(Flox)/WT lines (differentthan in FIG. 1) following Cre deletion or control GFP treatment, carriedout in triplicate. FIG. 6B shows average of 5 independentex4^(Flox)/S1KO and 2 independent ex4^(Flox)/WT lines. FIG. 6C showscolony formation assays of 5 independent ex4^(Flox)/S1KO and 2independent ex4^(Flox)/WT MEF lines, following GFP-Cre deletion or GFPcontrol treatment. Experiments were carried out in triplicate for eachMEF line FIG. 6D shows serial passaging of a second independent latepassage S1KO MEF line following reconstitution with retroviral SIRT1 atpassage 7 (arrow). FIG. 6E shows proliferation curves of SIRT1KO MEFsreconstituted with recombinant WT (SIRT1WT) or mutant SIRT1 (SIRT1HY)retrovirus at passage 5. Uninfected S1KO and WT MEFs are shown forcomparison. The data represent the average of three independentexperiments. Following selection for retrovirus, cells were seeded at10⁶ per well of 6-well plates, and harvested and counted each day. Cellnumbers were normalized to day 1. FIG. 6F shows colony formation assaysof four late passage S1KO lines following reconstitution with retroviralSIRT1 or empty virus control. p53-deficient MEFs and NIH3T3 lines areshown for comparison. In all panels, error bars represent the standarderror of the mean.

FIGS. 7A-7B show cell-cycle arrest in response to the indicated doses ofadriamycin (FIG. 7A) and ionizing radiation (FIG. 7B). For bothadriamycin and ionizing radiation experiments, cells were subjected to afour hour BrdU pulse 18 hours following treatment, and BrdUincorporation determined by flow cytometry. Data represent the mean ofthree independent cell lines and error bars indicate the standard errorof the mean.

FIGS. 8A-8C show oxidative stress measurements following culture inchronic sub-lethal oxidative conditions, and exposure to oncogenic Ras.FIG. 8A show levels of reactive oxygen species in cultures shown in FIG.3, grown under low serum (3%) or sub-lethal concentrations of H₂O₂ (50uM), measured by DCFDA fluorescence. Values represent mean fluorescenceof three independent S1KO or WT MEF lines, and are normalized to controlcultures. FIG. 8B shows increased levels of 8-oxoguanine in culturesshown in (FIG. 8A), measured by flow cytometry. Values represent meanfluorescence, and are normalized to control cultures. FIG. 8C showslevels of reactive oxygen species measured by DCFDA fluorescence,following exposure to H-Ras^(V12), as shown in FIG. 4. In FIG. 8A andFIG. 8C, ROS levels were measured using the probe DCFDA(5-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetylester), according to the manufacturer's instructions (Molecular Probes).Briefly, cells were incubated with 0.1 uM DCFDA for 1 hour, harvested,and DCFDA mean fluorescence measured by flow cytometry. In FIG. 8B,levels of 8-oxoguanine, were measured using the OxyDNA fluorometricassay kit (Calbiochem), and assayed by flow cytometry.

FIGS. 9A-9D show SIRT1-deficient MEFs retain intact responses top19^(ARF), p16^(INK4A), and c-myc. FIG. 9A shows proliferative arrestinduced by retroviral expression of p19^(ARF). FIG. 9B showsproliferative arrest induced by retroviral expression of p16^(INK4A).FIG. 9C shows sample soft agar assays of primary WT MEFs, late passageS1KO MEFs, and p53-deficient MEFs following expression of retroviralc-myc (gift of Dr. William Hahn). FIG. 9D show summary of soft agarassays of the indicated MEF lines following transduction with c-myc orcontrol retrovirus.

FIGS. 10A-D show genomic stability and telomere function. FIG. 10A showsmetaphase chromosome spreads and genomic instability assays. Chromosomespreads were obtained using standard protocols (Kaushal et al., 2003).Briefly, MEF cultures were held for 3 hours in media supplemented 100ng/ml colcemid to arrest cells in metaphase. Cells were dissociated andincubated in 75 mM KCl for 12-15 minutes before fixation in 3:1methanol:acetic acid overnight and preparation of slides. To assay forgenomic instability, metaphase chromosome spreads were stained with4′6′-diamidino-2-phenylindole hydrochloride (DAPI), photographed using aNikon E800 microscope equipped with a CCD camera, and scored on thepresence of structural chromosomal abnormalities. The data represent theaverage of three independent wild-type and S1KO MEF lines, and 20metaphases per line. FIG. 10B shows measurement of telomere restrictionfragment (TRF) length. P15 MEFs were embedded in agarose plugs (CleanCutAgarose, BioRad, Hercules, Calif.) and lysed in 2 mg/mL proteinase K at50° C. overnight. After Mbo I digestion, restriction fragments wereresolved at 6 V/cm² for 23 hr using a pulsed field electrophoresischamber (CHEF Mapper, Biorad), blotted to a nylon membrane andhybridized with a γ³²P-labeled 800 bp telomere (TTAGGG (SEQ ID. NO:1))probe (gift of T. de Lange, Rockefeller University, New York). FIG. 10Cshows telomere fluorescence in situ hybridization (FISH). P20 MEFs wereincubated in colcemid (KaryoMAX, GibcoBRL), swollen in 30 mM sodiumcitrate, fixed in methanol/acetic acid (3/1) and air dried on slidesovernight. After pepsin digestion, slides were denatured at 80° C. for 3min, hybridized with a Cy3-labeled PNA telomeric probe (Cy3-(TTAGGG)₃(SEQ ID. NO:2); Applied Biosystems, Bedford, Mass.) in 70% formamide atRT for 2 hr, washed, dehydrated, and mounted in Vectashield with DAPI(Vector Laboratories, Burlingame, Calif.). Images were taken with aNikon Eclipse microscope equipped with a CCD camera (Applied SpectralImaging, Carlsbad, Calif.) and a 63× objective lens. ˜30 metaphases ofeach genotype were scored for total structural aberrations (chromatidbreaks, detached centromeres, translocations) and chromosomalaberrations involving telomeres, such as “signal-free ends”, end-to-endchromosomal fusions, rings, or the presence of extrachromosomaltelomeric DNA, as summarized in FIG. 10D.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to the discovery that mouse embryonic fibroblasts(MEFs) deficient for SIRT1, a mammalian Sir2 homolog, have dramaticallyincreased resistance to replicative senescence. Extended replicativelifespan of SIRT1-deficient MEFs is associated with enhancedproliferative capacity under conditions of chronic, sublethal oxidativestress. Such SIRT1-deficient cells show normal p19ARF induction andcell-cycle arrest in response to DNA damage or oncogene expression.These results demonstrate that SIRT1 functions to promote replicativesenescence.

Accordingly, the present invention provides methods to increase thereplicative capacity of mammalian cells, by culturing cells in thepresence of an effective amount of a compound or agent that suppressesthe production or activity of SIRT1. Preferably, the compound interfereswith the expression of the SIRT1 gene. Such compounds include, forexample, siRNAs, antisense oligonucleotides, ribozymes, RNAi, andantibodies.

The expanded cultured cells of the invention can be used for a varietyof applications, including cell-based therapies, sometimes referred toas cell replacement therapies, such as bone marrow transplants, genetherapies, tissue engineering, and in vitro organogenesis.

As used herein, the cells cultured in the presence of the SIRT1inhibitor of the invention and expanded ex vivo are sometimes referredto as the cultured cells, or the expanded cells, or the expandedcultured cells.

Methods for Cell Expansion

Any compound or agent which inhibits SIRT1 can be used in the methods ofthe invention. In one embodiment, the agent inhibits the activity of theSIRT1. In another embodiment, the agent or compound inhibits SIRT1 bydecreasing transcription. Preferred inhibitors include DNA, RNA, an RNAinterfering agent, PNA, a small organic molecule, a natural product, aprotein, an antibody, a peptide and a peptidomimetic.

Inhibition of SIRT1 Activity

One preferred embodiment of the invention provides a method forenhancing the replicative lifespan of cells by using an agent to inhibitSIRT1, wherein the agent or compound inhibits the activity of the SIRT1.This can be accomplished by a range of different approaches, includingthe use of antibodies, small molecules, and antagonists.

Any agent which inhibits the activity of SIRT1 can be used in thepresent invention. For example, one class of preferred compounds aresirtuin inhibitors, including but not limited to the sirtuin inhibitorsdisclosed in Grozinger et al., J. Biol. Chem. 42:38837-43 (2001), whichis hereby incorporated by reference in its entirety. Preferred sirtuininhibitors include the compounds A3, sirtinol, and M15 describedtherein. In one embodiment, sirtinol is a particularly preferredinhibitor.

Another preferred method of inhibiting SIRT1 provides a peptide whichwould competitively bind and thus inhibit SIRT1.

Further optimization of effective dosages can be determined empiricallybased on the specific tissue and cell type.

Downregulation of SIRT1

Preferably, SIRT1 expression may be inhibited ex vivo by the use of anymethod which results in decreased transcription of the gene encodingSIRT1. The sequence of the gene encoding mouse SIRT1 is available inGenbank asgenomic contig accession number NT 039495 (Mus musculuschromosome 10 genomic contig). The accession number for the geneencoding mouse SIRT1 is available in Genbank at NM_(—)019812 (Musmusculus sirtuin 1). The sequence of the gene encoding human isavailable in Genbank as accession number NM 012238 (Homo sapiens sirtuin(silent mating type information regulation 2 homolog) 1), (SIRT1), mRNA;the DNA sequence of the human sequence from clone RP11-57G10 onchromosome 10 which includes the SIRT1 gene is available in Genbank asaccession number AL133551.

In one preferred embodiment, RNAi technology can be used to inhibit ordownregulate the expression of SIRT1 by decreasing transcription of thegene encoding SIRT1. RNA interference or “RNAi” is a term initiallycoined by Fire and co-workers to describe the observation thatdouble-stranded RNA (dsRNA) can block gene expression when it isintroduced into worms (Fire et al. (1998) Nature 391, 806-811). “RNAinterference (RNAi)” is an evolutionally conserved process whereby theexpression or introduction of RNA of a sequence that is identical orhighly similar to a target gene results in the sequence specificdegradation or specific post-transcriptional gene silencing (PTGS) ofmessenger RNA (mRNA) transcribed from that targeted gene (see Coburn, G.and Cullen, B. (2002) J. of Virology 76(18):9225), thereby inhibitingexpression of the target gene. In one embodiment, the RNA is doublestranded RNA (dsRNA). This process has been described in plants,invertebrates, and mammalian cells. In nature, RNAi is initiated by thedsRNA-specific endonuclease Dicer, which promotes processive cleavage oflong dsRNA into double-stranded fragments termed siRNAs. siRNAs areincorporated into a protein complex that recognizes and cleaves targetmRNAs. RNAi can also be initiated by introducing nucleic acid molecules,e.g., synthetic siRNAs or RNA interfering agents, to inhibit or silencethe expression of target genes. See for example U.S. patent applicationSer. Nos: 20030153519A1; 20030167490A1; and U.S. Pat. Nos. 6,506,559;6,573,099, which are herein incorporated by reference in their entirety.

Isolated RNA molecules specific to SIRT1 mRNA, which mediate RNAi, areantagonists useful in the method of the present invention. In oneembodiment, the RNA interfering agents used in the methods of theinvention, e.g., the siRNAs used in the methods of the invention, can tobe taken up actively by cells ex vivo by their addition to the culturemedium, illustrating efficient delivery of the RNA interfering agents,e.g., the siRNAs used in the methods of the invention.

Other strategies for delivery of the RNA interfering agents, e.g., thesiRNAs or shRNAs of used in the methods of the invention, may also beemployed, such as, for example, delivery by a vector, e.g., a plasmid orviral vector, e.g., a lentiviral vector. Other delivery methods includedelivery of the RNA interfering agents, e.g., the siRNAs or shRNAs ofthe invention, using a basic peptide by conjugating or mixing the RNAinterfering agent with a basic peptide, e.g., a fragment of a TATpeptide, mixing with cationic lipids or formulating into particles.

The RNA interfering agents, e.g., the siRNAs of the invention, may bedelivered singly, or in combination with other RNA interfering agents,e.g., siRNAs, such as, for example siRNAs directed to other cellulargenes, e.g., apoptosis-related genes. The RNA interfering agents, e.g.,siRNAs of the invention may also be administered in combination withother pharmaceutical agents which are used to treat or prevent cancer.

An “RNA interfering agent” as used herein, is defined as any agent whichinterferes with or inhibits expression of a target gene or genomicsequence by RNA interference (RNAi). Such RNA interfering agentsinclude, but are not limited to, nucleic acid molecules including RNAmolecules which are homologous to the target gene or genomic sequence,or a fragment thereof, short interfering RNA (siRNA), short hairpin orsmall hairpin RNA (shRNA), and small molecules which interfere with orinhibit expression of a target gene by RNA interference (RNAi). Thetarget gene of the present invention is the gene encoding SIRT1.

As used herein, “inhibition of target gene expression” includes anydecrease in expression or protein activity or level of the target geneor protein encoded by the target gene. The decrease may be of at least30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or more as compared to theexpression of a target gene or the activity or level of the proteinencoded by a target gene which has not been targeted by an RNAinterfering agent.

“Short interfering RNA” (siRNA), also referred to herein as “smallinterfering RNA” is defined as an agent which functions to inhibitexpression of a target gene, e.g., by RNAi. An siRNA may be chemicallysynthesized, may be produced by in vitro transcription, or may beproduced within a host cell. In one embodiment, siRNA is a doublestranded RNA (dsRNA) molecule of about 15 to about 40 nucleotides inlength, preferably about 15 to about 28 nucleotides, more preferablyabout 19 to about 25 nucleotides in length, and more preferably about19, 20, 21, or 22 nucleotides in length, and may contain a 3′ and/or 5′overhang on each strand having a length of about 0, 1, 2, 3, 4, 5, or 6nucleotides. The length of the overhang is independent between the twostrands, i.e., the length of the overhang on one strand is not dependenton the length of the overhang on the second strand. In one embodiment,the siRNA can inhibit SIRT1s by transcriptional silencing. Preferablythe siRNA is capable of promoting RNA interference through degradationor specific post-transcriptional gene silencing (PTGS) of the targetmessenger RNA (mRNA).

siRNAs also include small hairpin (also called stem loop) RNAs (shRNAs).In one embodiment, these shRNAs are composed of a short (e.g., about 19to about 25 nucleotide) antisense strand, followed by a nucleotide loopof about 5 to about 9 nucleotides, and the analogous sense strand.Alternatively, the sense strand may precede the nucleotide loopstructure and the antisense strand may follow. These shRNAs may becontained in plasmids, retroviruses, and lentiviruses and expressedfrom, for example, the pol III U6 promoter, or another promoter (see,e.g., Stewart, et al. (2003) RNA April; 9(4):493-501, incorporated bereference herein).

An siRNA may be substantially homologous to the target SIRT1 gene orgenomic sequence, or a fragment thereof. As used herein, the term“homologous” is defined as being substantially identical, sufficientlycomplementary, or similar to the target mRNA, or a fragment thereof, toeffect RNA interference of the target. In addition to native RNAmolecules, RNAs suitable for inhibiting or interfering with theexpression of a target sequence include RNA derivatives and analogs.siRNA molecules need not be limited to those molecules containing onlyRNA, but, for example, further encompasses chemically modifiednucleotides and non-nucleotides, and also include molecules wherein aribose sugar molecule is substituted for another sugar molecule or amolecule which performs a similar function. Moreover, a non-naturallinkage between nucleotide residues may be used, such as aphosphorothioate linkage. The RNA strand can be derivatized with areactive functional group of a reporter group, such as a fluorophore.Particularly useful derivatives are modified at a terminus or termini ofan RNA strand, typically the 3′ terminus of the sense strand. Forexample, the 2′-hydroxyl at the 3′ terminus can be readily andselectively derivatizes with a variety of groups.

Other useful RNA derivatives incorporate nucleotides having modifiedcarbohydrate moieties, such as 2′O-alkylated residues or 2′-O-methylribosyl derivatives and 2′-O-fluoro ribosyl derivatives. The RNA basesmay also be modified. Any modified base useful for inhibiting orinterfering with the expression of a target sequence may be used. Forexample, halogenated bases, such as 5-bromouracil and 5-iodouracil canbe incorporated. The bases may also be alkylated, for example,7-methylguanosine can be incorporated in place of a guanosine residue.Non-natural bases that yield successful inhibition can also beincorporated. In a preferred embodiment, the RNA is stabilized byincluding purine nucleotides, such as adenosine or guanosinenucleotides. Alternatively, substitution of pyrimidine nucleotides bymodified analogues, e.g., substitution of uridine 2 nucleotide 3′overhangs by 2′-deoxythymidine is tolerated and does not affect theefficiency of RNAi. The absence of a 2′ hydroxyl significantly enhancesthe nuclease resistance of the overhang in tissue culture medium.

SIRT1 expression may also be inhibited in vivo by the use of any methodwhich results in decreased transcription of the gene encoding SIRT1. Oneembodiment uses antisense technology. Gene expression can be controlledthrough triple-helix formation or antisense DNA or RNA, both of whichmethods are based on binding of a polynucleotide to DNA or RNA. Anantisense nucleic acid molecule which is complementary to a nucleic acidmolecule encoding SIRT1 can be designed based upon the isolated nucleicacid molecules encoding SIRT1 by means known to those in the art.

Design and Preparation of siRNA Molecules

Synthetic siRNA molecules, including shRNA molecules, of the presentinvention can be obtained using a number of techniques known to those ofskill in the art. One preferred siRNA is described in detail below inExample 1. For example, the siRNA molecule can be chemically synthesizedor recombinantly produced using methods known in the art, such as usingappropriately protected ribonucleoside phosphoramidites and aconventional DNA/RNA synthesizer (see, e.g., Elbashir, S. M. et al.(2001) Nature 411:494-498; Elbashir, S. M., W. Lendeckel and T. Tuschl(2001) Genes & Development 15:188-200; Harborth, J. et al. (2001) J.Cell Science 114:4557-4565; Masters, J. R. et al. (2001) Proc. Natl.Acad. Sci., USA 98:8012-8017; and Tuschl, T. et al. (1999) Genes &Development 13:3191-3197). Alternatively, several commercial RNAsynthesis suppliers are available including, but not limited to, Proligo(Hamburg, Germany), Dharmacon Research (Lafayette, Colo., USA), PierceChemical (part of Perbio Science, Rockford, Ill., USA), Glen Research(Sterling, Va., USA), ChemGenes (Ashland, Mass., USA), and Cruachem(Glasgow, UK). As such, siRNA molecules are not overly difficult tosynthesize and are readily provided in a quality suitable for RNAi. Inaddition, dsRNAs can be expressed as stem loop structures encoded byplasmid vectors, retroviruses and lentiviruses (Paddison, P. J. et al.(2002) Genes Dev. 16:948-958; McManus, M. T. et al. (2002) RNA8:842-850; Paul, C. P. et al. (2002) Nat. Biotechnol. 20:505-508;Miyagishi, M. et al. (2002) Nat. Biotechnol. 20:497-500; Sui, G. et al.(2002) Proc. Natl. Acad. Sci., USA 99:5515-5520; Brummelkamp, T. et al.(2002) Cancer Cell 2:243; Lee, N. S., et al. (2002) Nat. Biotechnol.20:500-505; Yu, J. Y., et al. (2002) Proc. Natl. Acad. Sci., USA99:6047-6052; Zeng, Y., et al. (2002) Mol. Cell 9:1327-1333; Rubinson,D. A., et al. (2003) Nat. Genet. 33:401-406; Stewart, S. A., et al.(2003) RNA 9:493-501). These vectors generally have a polIII promoterupstream of the dsRNA and can express sense and antisense RNA strandsseparately and/or as a hairpin structures. Within cells, Dicer processesthe short hairpin RNA (shRNA) into effective siRNA.

The targeted region of the siRNA molecule of the present invention canbe selected from a given target gene sequence, e.g., anapoptosis-related gene or a cytokine, beginning from about 25 to 50nucleotides, from about 50 to 75 nucleotides, or from about 75 to 100nucleotides downstream of the start codon. Nucleotide sequences maycontain 5′ or 3′ UTRs and regions nearby the start codon. One method ofdesigning a siRNA molecule of the present invention involves identifyingthe 23 nucleotide sequence motif AA(N19)TT (where N can be anynucleotide) (SEQ ID NO:3) and selecting hits with at least 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75% G/C content. The “TT”portion of the sequence is optional. Alternatively, if no such sequenceis found, the search may be extended using the motif NA(N21) (SEQ IDNO:4), where N can be any nucleotide. In this situation, the 3′ end ofthe sense siRNA may be converted to TT to allow for the generation of asymmetric duplex with respect to the sequence composition of the senseand antisense 3′ overhangs. The antisense siRNA molecule may then besynthesized as the complement to nucleotide positions 1 to 21 of the 23nucleotide sequence motif. The use of symmetric 3′ TT overhangs may beadvantageous to ensure that the small interfering ribonucleoproteinparticles (siRNPs) are formed with approximately equal ratios of senseand antisense target RNA-cleaving siRNPs (Elbashir et al. (2001) supraand Elbashir et al. 2001 supra). Analysis of sequence databases,including but not limited to the NCBI, BLAST, Derwent and GenSeq as wellas commercially available oligosynthesis companies such as Oligoengine®,may also be used to select siRNA sequences against EST libraries toensure that only one gene is targeted.

Delivery of RNA Interfering Agents

Methods of delivering RNA interfering agents, e.g., an siRNA of thepresent invention, or vectors containing an RNA interfering agent, e.g.,an siRNA of the present invention, to the target cells, e.g., stemcells, for uptake include injection of a composition containing the RNAinterfering agent, e.g., an siRNA, or directly contacting the cell,e.g., a stem cell, with a composition comprising an RNA interferingagent, e.g., an siRNA.

A viral-mediated delivery mechanism may also be employed to deliversiRNAs to cells in vitro and in vivo as described in Xia, H. et al.(2002) Nat Biotechnol 20(10): 1006). Plasmid- or viral-mediated deliverymechanisms of shRNA may also be employed to deliver shRNAs to cells invitro and in vivo as described in Rubinson, D. A., et al. ((2003) Nat.Genet. 33:401-406) and Stewart, S. A., et al. ((2003) RNA 9:493-501).Other methods of introducing siRNA molecules of the present invention totarget cells, e.g., tumor cells, include a variety of art-recognizedtechniques including, but not limited to, calcium phosphate or calciumchloride co-precipitation, DEAE-dextran-mediated transfection,lipofection, or electroporation as well as a number of commerciallyavailable transfection kits (e.g., OLIGOFECTAMINE® Reagent fromInvitrogen) (see, e.g. Sui, G. et al. (2002) Proc. Natl. Acad. Sci. USA99:5515-5520; Calegari, F. et al. (2002) Proc. Natl. Acad. Sci., USAOct. 21, 2002; J-M Jacque, K. Triques and M. Stevenson (2002) Nature418:435-437; and Elbashir, S. M et al. (2001) supra). Suitable methodsfor transfecting a target cell, e.g., a tubular cell of the kidney or acardiac cell can also be found in Sambrook, et al. (Molecular Cloning: ALaboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and otherlaboratory manuals. The efficiency of transfection may depend on anumber of factors, including the cell type, the passage number, theconfluency of the cells as well as the time and the manner of formationof siRNA- or shRNA-liposome complexes (e.g., inversion versusvortexing). These factors can be assessed and adjusted without undueexperimentation by one with ordinary skill in the art.

The RNA interfering agents, e.g., the siRNAs or shRNAs of the invention,may be introduced along with components that perform one or more of thefollowing activities: enhance uptake of the RNA interfering agents,e.g., siRNA, by the cell, e.g., tumor cells, inhibit annealing of singlestrands, stabilize single strands, or otherwise facilitate delivery tothe target cell and increase inhibition of the target gene, SIRT1.

Cells

The methods of the present invention can be used to enhance thereplicative lifespan of any cells which can be cultured. The methods ofthe present invention can be used to increase the replicative lifespanof any cells for which it is desirable to expand cells in vitro,including stem cells and non-stem cells. Preferred cells includeembryonic stem cells, somatic stem cells, umbilical cord blood stemcells, unrestricted somatic stem cells (USSC) derived from humanumbilical cord blood, postpartum-derived cells, mesenchymal stem cells,mesenchymal progenitor cells, hematopoietic lineage stem cells,hematopoietic lineage progenitor cells, neural stem cells, neuralprogenitor cells, endothelial stem cells, endothelial progenitor cells,and fibroblasts. Preferred somatic stem cells include bone marrowderived stem cells, adipose derived stem cells, mesenchymal stem cells,neural stem cells, liver stem cells, hepatocyte precursor cells,pancreatic stem cells, skin stem cells, and corneal epithelium stemcells.

One preferred embodiment of the invention provides methods to expandsomatic stem cells from the skin, to generate new tissue for use in skingrafts. Another preferred embodiment of the invention provides methodsto expand hematopoeitic stem cells or hair stem cells (Blainpain et al,2004). Yet another preferred embodiment of the invention providesmethods to expand hepatocytes and hepatocyte precursor cells (Mikula etal., 2004). The invention also provides methods to expand cartilagecells.

In one preferred embodiment, the cells are human cells. In anotherpreferred embodiment, the cells are murine cells.

In one preferred embodiment, the cultured cells are capable ofself-renewal and expansion in culture, and have the potential todifferentiate into cells of other phenotypes.

In one aspect, the invention provides postpartum-derived cells (PPDCs)derived from postpartum tissue substantially free of blood. The PPDCsmay be derived from placenta of a mammal including but not limited tohuman. The cells are capable of self-renewal and expansion in culture.The postpartum-derived cells have the potential to differentiate intocells of other phenotypes. The invention provides, in one of its severalaspects cells that are derived from umbilical cord, as opposed toumbilical cord blood. The invention also provides, in one of its severalaspects, cells that are derived from placental tissue.

In one embodiment, somatic tissue stem cells of the present inventioninclude any stem cells isolated from adult tissue. Somatic stem cellsinclude but are not limited to bone marrow derived stem cells, adiposederived stem cells, mesenchymal stem cells, neural stem cells, liverstem cells, and pancreatic stem cells. Bone marrow derived stem cellsrefers to all stem cells derived from bone marrow; these include but arenot limited to mesenchymal stem cells, bone marrow stromal cells, andhematopoietic stem cells. Bone marrow stem cells are also known asmesenchymal stem cells or bone marrow stromal stem cells, or simplystromal cells or stem cells.

In certain embodiments, the stem cells act as precursor cells, whichproduce daughter cells that mature into differentiated cells. The stemcells can be isolated from the individual in need of stem cell therapyor from another individual. Preferably, the individual is a matchedindividual to insure that rejection problems do not occur. Therapies toavoid rejection of foreign cells are known in the art. Furthermore,somatic stem cells may be immune-privileged, so the graft versus hostdisease after allogenic transplant may be minimal or non-existent(Weissman, 2000). Endogenous or stem cells from a matched donor may beadministered by any known means, preferably intravenous injection, orinjection directly into the appropriate tissue.

In some embodiments, somatic tissue stem cells can be isolated fromfresh bone marrow or adipose tissue by fractionation using fluorescenceactivated call sorting (FACS) with unique cell surface antigens toisolate specific subtypes of stem cells (such as bone marrow or adiposederived stem cells) for injection into recipients following expansion invitro, as described above.

As stated above, cells may be derived from the individual to be treatedor a matched donor. Those having ordinary skill in the art can readilyidentify matched donors using standard techniques and criteria.

Two preferred embodiments provide bone marrow or adipose tissue derivedstem cells, which may be obtained by removing bone marrow cells or fatcells, from a donor, either self or matched, and placing the cells in asterile container with a plastic surface or other appropriate surfacethat the cells come into contact with. The stromal cells will adhere tothe plastic surface within 30 minutes to about 6 hours. After at least30 minutes, preferably about four hours, the non-adhered cells may beremoved and discarded. The adhered cells are stem cells, which areinitially non-dividing. After about 2-4 days however the cells begin toproliferate.

Stem cells are undifferentiated cells defined by their ability at thesingle cell level to both self-renew and differentiate to produceprogeny cells, including self-renewing progenitors, non-renewingprogenitors and terminally differentiated cells. Stem cells are alsocharacterized by their ability to differentiate in vitro into functionalcells of various cell lineages from multiple germ layers (endoderm,mesoderm and ectoderm), as well as to give rise to tissues of multiplegerm layers following transplantation and to contribute substantially tomost, if not all, tissues following injection into blastocysts.

Stem cells are classified by their developmental potential as: (1)totipotent—able to give rise to all embryonic and extraembryonic celltypes; (2) pluripotent—able to give rise to all embryonic cell types;(3) multipotent—able to give rise to a subset of cell lineages, but allwithin a particular tissue, organ, or physiological system (for example,hematopoietic stem cells (HSC) can produce progeny that include HSC(self-renewal), blood cell-restricted oligopotent progenitors, and allcell types and elements (e.g., platelets) that are normal components ofthe blood); (4) oligopotent—able to give rise to a more restrictedsubset of cell lineages than multipotent stem cells; and (5)unipotent—able to give rise to a single cell lineage (e.g.,spermatogenic stem cells).

Stem cells are also categorized on the basis of the source from whichthey may be obtained. An adult stem cell is generally a multipotentundifferentiated cell found in tissue comprising multiple differentiatedcell types. The adult stem cell can renew itself and, under normalcircumstances, differentiate to yield the specialized cell types of thetissue from which it originated, and possibly other tissue types. Anembryonic stem cell is a pluripotent cell from the inner cell mass of ablastocyst-stage embryo. A fetal stem cell is one that originates fromfetal tissues or membranes. A postpartum stem cell is a multipotent orpluripotent cell that originates substantially from extraembryonictissue available after birth, namely, the placenta and the umbilicalcord. These cells have been found to possess features characteristic ofpluripotent stem cells, including rapid proliferation and the potentialfor differentiation into many cell lineages. Postpartum stem cells maybe blood-derived (e.g., as are those obtained from umbilical cord blood)or non-blood-derived (e.g., as obtained from the non-blood tissues ofthe umbilical cord and placenta).

Embryonic tissue is typically defined as tissue originating from theembryo (which in humans refers to the period from fertilization to aboutsix weeks of development. Fetal tissue refers to tissue originating fromthe fetus, which in humans refers to the period from about six weeks ofdevelopment to parturition. Extraembryonic tissue is tissue associatedwith, but not originating from, the embryo or fetus. Extraembryonictissues include extraembryonic membranes (chorion, amnion, yolk sac andallantois), umbilical cord and placenta (which itself forms from thechorion and the maternal decidua basalis).

Differentiation is the process by which an unspecialized (“uncommitted”)or less specialized cell acquires the features of a specialized cell,such as a nerve cell or a muscle cell, for example. A differentiated ordifferentiation-induced cell is one that has taken on a more specialized(“committed”) position within the lineage of a cell. The term committed,when applied to the process of differentiation, refers to a cell thathas proceeded in the differentiation pathway to a point where, undernormal circumstances, it will continue to differentiate into a specificcell type or subset of cell types, and cannot, under normalcircumstances, differentiate into a different cell type or revert to aless differentiated cell type. De-differentiation refers to the processby which a cell reverts to a less specialized (or committed) positionwithin the lineage of a cell. As used herein, the lineage of a celldefines the heredity of the cell, i.e., which cells it came from andwhat cells it can give rise to. The lineage of a cell places the cellwithin a hereditary scheme of development and differentiation. Alineage-specific marker refers to a characteristic specificallyassociated with the phenotype of cells of a lineage of interest and canbe used to assess the differentiation of an uncommitted cell to thelineage of interest.

In a broad sense, a progenitor cell is a cell that has the capacity tocreate progeny that are more differentiated than itself and yet retainsthe capacity to replenish the pool of progenitors. By that definition,stem cells themselves are also progenitor cells, as are the moreimmediate precursors to terminally differentiated cells. When referringto the cells of the present invention, as described in greater detailbelow, this broad definition of progenitor cell may be used. In anarrower sense, a progenitor cell is often defined as a cell that isintermediate in the differentiation pathway, i.e., it arises from a stemcell and is intermediate in the production of a mature cell type orsubset of cell types. This type of progenitor cell is generally not ableto self-renew. Accordingly, if this type of cell is referred to herein,it will be referred to as a non-renewing progenitor cell or as anintermediate progenitor or precursor cell.

As used herein, the phrase differentiates into a mesodermal, ectodermalor endodermal lineage refers to a cell that becomes committed to aspecific mesodermal, ectodermal or endodermal lineage, respectively.Examples of cells that differentiate into a mesodermal lineage or giverise to specific mesodermal cells include, but are not limited to, cellsthat are adipogenic, chondrogenic, cardiogenic, dermatogenic,hematopoetic, hemangiogenic, myogenic, nephrogenic, urogenitogenic,osteogenic, pericardiogenic, or stromal. Examples of cells thatdifferentiate into ectodermal lineage include, but are not limited toepidermal cells, neurogenic cells, and neurogliagenic cells. Examples ofcells that differentiate into endodermal lineage include, but are notlimited to pleurigenic cells, and hepatogenic cells, cell that give riseto the lining of the intestine, and cells that give rise to pancreogenicand splanchogenic cells.

One preferred cell of the invention is postpartum-derived cells (PPDCs).Subsets of the cells of the present invention are referred to asplacenta-derived cells (PDCs) or umbilical cord-derived cells (UDCs).PPDCs of the invention encompass undifferentiated anddifferentiation-induced cells. In addition, the cells may be describedas being stem or progenitor cells, the latter term being used in thebroad sense. The term derived is used to indicate that the cells havebeen obtained from their biological source and grown or otherwisemanipulated in vitro (e.g., cultured in a growth medium to expand thepopulation and/or to produce a cell line).

Various terms are used to describe cells in culture. Cell culture refersgenerally to cells taken from a living organism and grown undercontrolled condition (“in culture”). A primary cell culture is a cultureof cells, tissues or organs taken directly from organisms and before thefirst subculture. Cells are expanded in culture when they are placed ina growth medium under conditions that facilitate cell growth and/ordivision, resulting in a larger population of the cells. When cells areexpanded in culture, the rate of cell proliferation is sometimesmeasured by the amount of time needed for the cells to double in number.This is referred to as doubling time.

A cell line is a population of cells formed by one or moresubcultivations of a primary cell culture. Each round of subculturing isreferred to as a passage. When cells are subcultured, they are referredto as having been passaged. A specific population of cells, or a cellline, is sometimes referred to or characterized by the number of timesit has been passaged. For example, a cultured cell population that hasbeen passaged ten times may be referred to as a P10 culture. The primaryculture, i.e., the first culture following the isolation of cells fromtissue, is designated P0. Following the first subculture, the cells aredescribed as a secondary culture (P1 or passage 1). After the secondsubculture, the cells become a tertiary culture (P2 or passage 2), andso on. It will be understood by those of skill in the art that there maybe many population doublings during the period of passaging; thereforethe number of population doublings of a culture is greater than thepassage number. The expansion of cells (i.e., the number of populationdoublings) during the period between passaging depends on many factors,including but not limited to the seeding density, substrate, medium, andtime between passaging.

When referring to cultured vertebrate cells, the term senescence (alsoreplicative senescence or cellular senescence) refers to a propertyattributable to finite cell cultures; namely, their inability to growbeyond a finite number of population doublings (sometimes referred to asHayflick's limit). Although cellular senescence was first describedusing fibroblast-like cells, most normal human cell types that can begrown successfully in culture undergo cellular senescence. The in vitrolifespan of different cell types varies, but the maximum lifespan istypically fewer than 100 population doublings (this is the number ofdoublings for all the cells in the culture to become senescent and thusrender the culture unable to divide). Senescence does not depend onchronological time, but rather is measured by the number of celldivisions, or population doublings, the culture has undergone. Thus,cells made quiescent by removing essential growth factors are able toresume growth and division when the growth factors are re-introduced,and thereafter carry out the same number of doublings as equivalentcells grown continuously. Similarly, when cells are frozen in liquidnitrogen after various numbers of population doublings and then thawedand cultured, they undergo substantially the same number of doublings ascells maintained unfrozen in culture. Senescent cells are not dead ordying cells; they are actually resistant to programmed cell death(apoptosis), and have been maintained in their nondividing state for aslong as three years. These cells are very much alive and metabolicallyactive, but they do not divide.

Cell Culture Methods

The methods of the present invention enhance the replicative lifespan ofthe cells cultured in the presence of the SIRT1 inhibitor, resulting intheir expansion in vitro. Preferably, the cultured cells undergo atleast one mitotic cell division. Even more preferably, the culturedcells undergo at least ten mitotic cell divisions.

Cells can be obtained from donor tissue by dissociation of individualcells from the connecting extracellular matrix of the tissue. Tissue isremoved using a sterile procedure, and the cells are dissociated usingany method known in the art including treatment with enzymes such astrypsin, collagenase, and the like, or by using physical methods ofdissociation such as with a blunt instrument.

Dissociation of cells can be carried out in any acceptable medium,including tissue culture medium. For example, a preferred medium for thedissociation of neural stem cells is low calcium artificialcerebrospinal fluid. The dissociated cells can be placed into any knownculture medium capable of supporting cell growth, including HEM, DMEM,RPMI, F-12, and the like, containing supplements which are required forcellular metabolism such as glutamine and other amino acids, vitamins,minerals and useful proteins such as transferrin and the like. Mediummay also contain antibiotics to prevent contamination with yeast,bacteria and fungi such as penicillin, streptomycin, gentamicin and thelike. In some cases, the medium may contain serum derived from bovine,equine, chicken and the like. Serum can contain xanthine, hypoxanthine,or other compounds which enhance guanine nucleotide biosynthesis,although generally at levels below the effective concentration tosuppress asymmetric cell kinetics. Thus, preferably a defined,serum-free culture medium is used, as serum contains unknown components(i.e. is undefined). A defined culture medium is also preferred if thecells are to be used for transplantation purposes. A particularlypreferable culture medium is a defined culture medium comprising amixture of DMEM, F12, and a defined hormone and salt mixture.

The culture medium can be supplemented with a proliferation-inducinggrowth factor(s). As used herein, the term “growth factor” refers to aprotein, peptide or other molecule having a growth, proliferative,differentiative, or trophic effect on neural stem cells and/or neuralstem cell progeny. Growth factors that may be used include any trophicfactor that allows stem cells to proliferate, including any moleculethat binds to a receptor on the surface of the cell to exert a trophic,or growth-inducing effect on the cell. Preferred proliferation-inducinggrowth factors include EGF, amphiregulin, acidic fibroblast growthfactor (aFGF or FGF-1), basic fibroblast growth factor (bFGF or FGF-2),transforming growth factor alpha (TGF.alpha.), and combinations thereof.Growth factors are usually added to the culture medium at concentrationsranging between about 1 fg/ml to 1 mg/ml. Concentrations between about 1to 100 ng/ml are usually sufficient. Simple titration experiments can beeasily performed to determine the optimal concentration of a particulargrowth factor.

In addition to proliferation-inducing growth factors, other growthfactors may be added to the culture medium that influence proliferationand differentiation of the cells including NGF, platelet-derived growthfactor (PDGF), thyrotropin releasing hormone (TRH), transforming growthfactor betas (TGF.beta.s), insulin-like growth factor (IGF.sub.-1) andthe like.

Cells can be cultured in suspension or on a fixed substrate. Oneparticularly preferred substrate is a hydrogel, such as a peptidehydrogel, as described below. However, certain substrates tend to inducedifferentiation of certain cells. Thus, suspension cultures arepreferable for such cell populations. Cell suspensions can be seeded inany receptacle capable of sustaining cells, particularly culture flasks,cultures plates, or roller bottles, more particularly in small cultureflasks such as 25 cm² cultures flasks.

Conditions for culturing should be close to physiological conditions.The pH of the culture medium should be close to physiological pH,preferably between pH 6-8, more preferably between about pH 7 to 7.8,with pH 7.4 being most preferred. Physiological temperatures rangebetween about 30.degree. C. to 40.degree. C. Cells are preferablycultured at temperatures between about 32.degree. C. to about 38.degree.C., and more preferably between about 35.degree. C. to about 37.degree.C.

Cells are preferably cultured for 3-30 days, preferably at least about 7days, more preferably at least 10 days, still more preferably at leastabout 14 days. Cells can be cultured substantially longer. They can alsobe frozen using known methods such as cryopreservation, and thawed andused as needed.

Uses of Expanded Cultured Cells

The present invention also provides for the administration of expandedpopulations of cells to a patient in need thereof. The expanded cells ofthe present invention can be used for a variety of purposes, includingbut not limited to bone marrow transplants, gene therapies, tissueengineering, and in vitro organogenesis. Production of autologous cellsto replace injured tissue would also reduce the need for immunesuppression interventions.

Preferred tissues for the isolation and expansion of cells, foradministration to a patient in need thereof, include but are not limitedto the following: bone marrow, liver, lung, small intestine, colon, skinand cartilage such as from the knee.

One preferred embodiment of the invention provides administration ofexpanded skin cells to a patient in need thereof, includingadministration of new tissue for use in skin grafts. Another preferredembodiment of the invention provides administration of expandedhepatocytes to a patient in need thereof (Mikula et al, 2004). Yetanother preferred embodiment of the invention provides administration ofexpanded cartilage cells to a patient in need thereof.

Yet another preferred embodiment of the invention providesadministration of expanded hematopoeitic stem cells or hair stem cells(Blainpain et al, 2004).

Gene Therapy Applications

According to the invention, the cultured expanded cells can be furthergenetically altered prior to reintroducing the cells into the individualfor gene therapy, to introduce a gene whose expression has therapeuticeffect on the individual. Methods for introducing genes into thecultured cells are provided in detail below.

In some aspects of the invention, individuals can be treated bysupplementing, augmenting and/or replacing defective and/or damagedcells with cells that express a therapeutic gene. The cells may bederived from cells of a normal matched donor or stem cells from theindividual to be treated (i.e., autologous). By introducing normal genesin expressible form, individuals suffering from such a deficiency can beprovided the means to compensate for genetic defects and eliminate,alleviate or reduce some or all of the symptoms.

Administration of Expanded Cultured Cells

This method involves administering by standard means, such asintravenous infusion or mucosal injection, the expanded cultured cellsto a patient.

The discovery that cells may be expanded ex vivo and administeredintravenously provides the means for systemic administration. Forexample, bone marrow-derived stem cells may be isolated with relativeease and the isolated cells may be cultured according to methods of thepresent invention to increase the number of cells available. Intravenousadministration also affords ease, convenience and comfort at higherlevels than other modes of administration. In certain applications,systemic administration by intravenous infusion is more effectiveoverall. In a preferred embodiment, the stem cells are administered toan individual by infusion into the superior mesenteric artery or celiacartery. The cells may also be delivered locally by irrigation down therecipient's airway or by direct injection into the mucosa of theintestine.

After isolating the cells, the cells can cultured for a period of timesufficient to allow them to expand to desired numbers, without any lossof desired functional characteristics. For example cells can be culturedfrom 1 day to over a year. Preferably the cells are cultured for 3-30days, more preferably 4-14 days, most preferably at least 7 days.

In one embodiment of the invention, the cultured cells can be induced todifferentiate following expansion in vitro, prior to administration tothe individual.

Differentiation of the cultured cells can be induced by any method knownin the art which activates the cascade of biological events which leadto growth, which include the liberation of inositol triphosphate andintracellular Ca.²⁺, liberation of diacyl glycerol and the activation ofprotein kinase C and other cellular kinases, and the like. Treatmentwith phorbol esters, differentiation-inducing growth factors and otherchemical signals can induce differentiation. Differentiation can also beinduced by plating the cells on a fixed substrate such as flasks,plates, or coverslips coated with an ionically charged surface such aspoly-L-lysine and poly-L-ornithine and the like.

Other substrates may be used to induce differentiation such as collagen,fibronectin, laminin, MATRIGEL™ (Collaborative Research), and the like.Differentiation can also be induced by leaving the cells in suspensionin the presence of a proliferation-inducing growth factor, withoutreinitiation of proliferation.

A preferred method for inducing differentiation of certain stem cellscomprises culturing the cells on a fixed substrate in a culture mediumthat is free of the proliferation-inducing growth factor. After removalof the proliferation-inducing growth factor, the cells adhere to thesubstrate (e.g. poly-omithine-treated plastic or glass), flatten, andbegin to differentiate into neurons and glial cells. At this stage theculture medium may contain serum such as 0.5-1.0% fetal bovine serum(FBS). However, for certain uses, if defined conditions are required,serum would not be used.

Differentiation can be determined using immunocytochemistry techniqueswell known in the art. Immunocytochemistry (e.g. dual-labelimmunofluorescence and immunoperoxidase methods) utilizes antibodiesthat detect cell proteins to distinguish the cellular characteristics orphenotypic properties of differentiated cell types compared to markerspresent on stem cells.

For administration of the cultured cells, the cells can be removed fromculture dishes, washed with saline, centrifuged to a pellet andresuspended in a glucose solution which is infused into the patient.

Between 10⁵ and 10¹³ cells per 100 kg person are administered perinfusion. Preferably, between about 1-5×10⁸ and 1-5×10¹² cells areinfused intravenously per 100 kg person. More preferably, between about1×10⁹ and 5×10¹¹ cells are infused intravenously per 100 kg person. Forexample, dosages such as 4×10⁹ cells per 100 kg person and 2×10¹¹ cellscan be infused per 100 kg person. The cells can also be injecteddirectly into the intestinal mucosa through an endoscope.

In some embodiments, a single administration of cells is provided. Inother embodiments, multiple administrations are used. Multipleadministrations can be provided over periodic time periods such as aninitial treatment regime of 3-7 consecutive days, and then repeated atother times.

The term “animal” here denotes all mammalian animals, including human.It also includes an individual animal in all stages of development,including embryonic and fetal stages. A “transgenic” animal is anyanimal containing cells that bear genetic information received, directlyor indirectly, by deliberate genetic manipulation at the subcellularlevel, such as by microinjection or infection with recombinant virus.

The term treating (or treatment of) a condition refers to amelioratingthe effects of, or delaying, halting or reversing the progress of, ordelaying or preventing the onset of, a condition as defined herein.

The term effective amount refers to a concentration of a reagent orpharmaceutical composition, such as a growth factor, differentiationagent, trophic factor, cell population or other agent, that is effectivefor producing an intended result, including cell growth and/ordifferentiation in vitro or in vivo, or treatment of a bone or cartilagecondition as described herein. With respect to growth factors, aneffective amount may range from about 1 nanogram/milliliter to about 1microgram/milliliter. With respect to cells as administered to a patientin vivo, an effective amount may range from as few as several hundred orfewer to as many as several million or more. In specific embodiments, aneffective amount may range from 10.sup.3-10.sup.11. It will beappreciated that the number of cells to be administered will varydepending on the specifics of the disorder to be treated, including butnot limited to size or total volume/surface area to be treated, as wellas proximity of the site of administration to the location of the regionto be treated, among other factors familiar to the medicinal biologist.

The terms effective period (or time) and effective conditions refer to aperiod of time or other controllable conditions (e.g., temperature,humidity for in vitro methods), necessary or preferred for an agent orpharmaceutical composition to achieve its intended result.

The term patient or subject refers to animals, including mammals,preferably humans, who are treated with the pharmaceutical compositionsor in accordance with the methods described herein.

The term matrix as used herein refers to a support for the cells of theinvention, for example, a scaffold (e.g., VICRYL, PCL/PGA, or RAD16) orsupporting medium (e.g., hydrogel, extracellular membrane protein (e.g.,MATRIGEL (BD Discovery Labware, Bedford, Mass.)).

The term pharmaceutically acceptable carrier (or medium), which may beused interchangeably with the term biologically compatible carrier ormedium, refers to reagents, cells, compounds, materials, compositions,and/or dosage forms which are, within the scope of sound medicaljudgment, suitable for use in contact with the tissues of human beingsand animals without excessive toxicity, irritation, allergic response,or other complication commensurate with a reasonable benefit/risk ratio.As described in greater detail herein, pharmaceutically acceptablecarriers suitable for use in the present invention include liquids,semi-solid (e.g., gels) and solid materials (e.g., cell scaffolds). Asused herein, the term biodegradable describes the ability of a materialto be broken down (e.g., degraded, eroded, dissolved) in vivo. The termincludes degradation in vivo with or without elimination (e.g., byresorption) from the body. The semi-solid and solid materials may bedesigned to resist degradation within the body (non-biodegradable) orthey may be designed to degrade within the body (biodegradable,bioerodable). A biodegradable material may further be bioresorbable orbioabsorbable, i.e., it may be dissolved and absorbed into bodily fluids(water-soluble implants are one example), or degraded and ultimatelyeliminated from the body, either by conversion into other materials orby breakdown and elimination through natural pathways.

Several terms are used herein with respect to cell-based therapies, alsoknown as cell replacement therapy. The terms autologous transfer,autologous transplantation, autograft and the like refer to treatmentswherein the cell donor is also the recipient of the cell replacementtherapy. The terms allogeneic transfer, allogeneic transplantation,allograft and the like refer to treatments wherein the cell donor is ofthe same species as the recipient of the cell replacement therapy, butis not the same individual. A cell transfer in which the donor's cellshave been histocompatibly matched with a recipient is sometimes referredto as a syngeneic transfer. The terms xenogeneic transfer, xenogeneictransplantation, xenograft and the like refer to treatments wherein thecell donor is of a different species than the recipient of the cellreplacement therapy.

Genetic Manipulation of Cultured Cells

In another embodiment of the invention, any gene(s) of interest can beintroduced into the culture cells. As explained below, it is preferredthat the genes are operably linked to an inducible promoter.

As used herein, the introduction of DNA into a host cell is referred toas transduction, sometimes also known as transfection or infection.Cultured cells, such as stem cells, can be transduced ex vivo at highefficiency.

As used herein, the terms “transgene”, “heterologous gene”, “exogenousgenetic material”, “exogenous gene” and “nucleotide sequence encodingthe gene” are used interchangeably and meant to refer to genomic DNA,cDNA, synthetic DNA and RNA, mRNA and antisense DNA and RNA which isintroduced into the cultured cell. The exogenous genetic material may beheterologous or an additional copy or copies of genetic materialnormally found in the individual or animal. When cells are to be used asa component of a pharmaceutical composition in a method for treatinghuman diseases, conditions or disorders, the exogenous genetic materialthat is used to transform the cells may also encode proteins selected astherapeutics used to treat the individual and/or to make the cells moreamenable to transplantation.

An expression cassette can be created for expression of the gene thatleads to constitutive upregulation of guanine ribonucleotides. Such anexpression cassette can include regulatory elements such as a promoter,an initiation codon, a stop codon, and a polyadenylation signal. It isnecessary that these elements be operable in the cultured cells or incells that arise from the cultured cells after infusion into anindividual. Moreover, it is necessary that these elements be operablylinked to the nucleotide sequence that encodes the protein such that thenucleotide sequence can be expressed in the cultured cells and thus theprotein can be produced. Initiation codons and stop codons are generallyconsidered to be part of a nucleotide sequence that encodes the protein.

A variety of promoters can be used for expression of the transgene.Promoters that can be used to express the gene are well known in theart. Promoters include cytomegalovirus (CMV) intermediate earlypromoter, a viral LTR such as the Rous sarcoma virus LTR, HIV-LTR,HTLV-1 LTR, the simian virus 40 (SV40) early promoter, E. coli lac UV5promoter and the herpes simplex tk virus promoter. For example, one canuse a tissue specific promoter, i.e. a promoter that functions in sometissues but not in others. Such promoters include EF2 responsivepromoters, etc. Regulatable promoters are preferred. Such systemsinclude those using the lac repressor from E. coli as a transcriptionmodulator to regulate transcription from lac operator-bearing mammaliancell promoters [Brown, M. et al., Cell, 49:603-612 (1987)], those usingthe tetracycline repressor (tetR) [Gossen, M., and Bujard, H., Proc.Natl. Acad. Sci. USA 89:5547-5551 (1992); Yao, F. et al., Human GeneTherapy, 9:1939-1950 (1998); Shockelt, P., et al., Proc. Natl. Acad.Sci. USA, 92:6522-6526 (1995)]. Other systems include FK506 dimer, VP16or p65 using astradiol, RU486, diphenol murislerone or rapamycin [seeMiller and Vvhelan, supra at FIG. 2]. Inducible systems are availablefrom Invitrogen, Clontech and Ariad. Systems using a repressor with theoperon are preferred. Regulation of transgene expression in target cellsrepresents a critical aspect of gene therapy. For example, the lacrepressor from Escherichia coli can function as a transcriptionalmodulator to regulate transcription from lac operator-bearing mammaliancell promoters [M. Brown et al., Cell, 49:603-612 (1987)]; Gossen andBujard (1992); [M. Gossen et al., Natl. Acad. Sci. USA, 89:5547-5551(1992)] combined the tetracycline repressor (tetR) with thetranscription activator (VP16) to create a tetR-mammalian celltranscription activator fusion protein, tTa (tetR-VP16), with theteto-bearing minimal promoter derived from the human cytomegalovirus(hCMV) major immediate-early promoter to create a tetR-tet operatorsystem to control gene expression in mammalian cells. Recently Yao andcolleagues [F. Yao et al., Human Gene Therapy, supra] demonstrated thatthe tetracycline repressor (tetR) alone, rather than the tetR-mammaliancell transcription factor fusion derivatives can function as potenttrans-modulator to regulate gene expression in mammalian cells when thetetracycline operator is properly positioned downstream for the TATAelement of the CMVIE promoter. One particular advantage of thistetracycline inducible switch is that it does not require the use of atetracycline repressor-mammalian cells transactivator or repressorfusion protein, which in some instances can be toxic to cells [M. Gossenet al., Natl. Acad. Sci. USA, 89:5547-5551 (1992); P. Shockett et al.,Proc. Natl. Acad. Sci. USA, 92:6522-6526 (1995)], to achieve itsregulatable effects.

The effectiveness of some inducible promoters increases over time. Insuch cases one can enhance the effectiveness of such systems byinserting multiple repressors in tandem, e.g. TetR linked to a TetR byan IRES. Alternatively, one can wait at least 3 days before screeningfor the desired function. While some silencing may occur, it isminimized given the large number of cells being used, preferably atleast 1×10⁴, more preferably at least 1×10⁵, still more preferably atleast 1×10⁶, and even more preferably at least 1×10⁷, the effect ofsilencing is minimal. One can enhance expression of desired proteins byknown means to enhance the effectiveness of this system. For example,using the Woodchuck Hepatitis Virus Posttranscriptional RegulatoryElement (WPRE). See Loeb, V. E., et al., Human Gene Therapy 10:2295-2305(1999); Zufferey, R., et al., J. of Virol. 73:2886-2892 (1999); Donello,J. E., et al., J. of Virol. 72:5085-5092 (1998).

Examples of polyadenylation signals useful to practice the presentinvention include but are not limited to human collagen Ipolyadenylation signal, human collagen II polyadenylation signal, andSV40 polyadenylation signal.

In order to maximize protein production, codons may be selected whichare most efficiently translated in the cell. The skilled artisan canprepare such sequences using known techniques based upon the presentdisclosure.

The exogenous genetic material that includes the transgene operablylinked to the regulatory elements may remain present in the cell as afunctioning cytoplasmic molecule, a functioning episomal molecule or itmay integrate into the cell's chromosomal DNA. Exogenous geneticmaterial may be introduced into cells where it remains as separategenetic material in the form of a plasmid. Alternatively, linear DNA,which can integrate into the chromosome, may be introduced into thecell. When introducing DNA into the cell, reagents, which promote DNAintegration into chromosomes, may be added. DNA sequences, which areuseful to promote integration, may also be included in the DNA molecule.Alternatively, RNA may be introduced into the cell.

Selectable markers can be used to monitor uptake of the desired gene.These marker genes can be under the control of any promoter or aninducible promoter. These are well known in the art and include genesthat change the sensitivity of a cell to a stimulus such as a nutrient,an antibiotic, etc. Genes include those for neo, puro, tk, multiple drugresistance (MDR), etc. Other genes express proteins that can readily bescreened for such as green fluorescent protein (GFP), blue fluorescentprotein (BFP), luciferase, LacZ, nerve growth factor receptor (NGFR),etc.

For example, one can set up systems to screen cultured cellsautomatically for the marker. In this way one can rapidly selecttransduced cultured cells from non-transformed cells. For example, theresultant particles can be contacted with about one million cells. Evenat transduction rates of 10-15% one will obtain 100-150,000 cells. Anautomatic sorter that screens and selects cells displaying the marker,e.g. GFP, can be used in the present method.

Vectors include chemical conjugates, plasmids, phage, etc. The vectorscan be chromosomal, non-chromosomal or synthetic. Commercial expressionvectors are well known in the art, for example pcDNA 3.1, pcDNA4 HisMax,pACH, pMT4, PND, etc. Preferred vectors include viral vectors, fusionproteins and chemical conjugates. Retroviral vectors include Moloneymurine leukemia viruses and pseudotyped lentiviral vectors such as FIVor HIV cores with a heterologous envelope. Other vectors include poxvectors such as orthopox or avipox vectors, herpesvirus vectors such asa herpes simplex I virus (HSV) vector (Geller, A. I. et al., (1995), J.Neurochem, 64: 487; Lim, F., et al., (1995) in DNA Cloning: MammalianSystems, D. Glover, Ed., Oxford Univ. Press, Oxford England; Geller, A.I. et al. (1993), Proc Natl. Acad. Sci.: U.S.A. 90:7603; Geller, A. I.,et al., (1990) Proc Natl. Acad. Sci USA 87:1149), adenovirus vectors(LeGal LaSalle et al. (1993), Science, 259:988; Davidson, et al. (1993)Nat. Genet 3: 219; Yang, et al., (1995) J. Virol. 69: 2004) andadeno-associated virus vectors (Kaplitt, M. G., et al. (1994) Nat.Genet. 8: 148).

The introduction of the gene into the cultured cells can be by standardtechniques, e.g. infection, transfection, transduction ortransformation. Examples of modes of gene transfer include e.g., nakedDNA, CaPO₄ precipitation, DEAE dextran, electroporation, protoplastfusion, lipofection, cell microinjection, and viral vectors,adjuvant-assisted DNA, gene gun, catheters, etc.

The vectors are used to transduce the cultured cells ex vivo. One canrapidly select the transduced cells by screening for the marker.Thereafter, one can take the transduced cells and grow them under theappropriate conditions or insert those cells into a host animal.

In one embodiment, the information to be introduced into the cell ispreferably foreign to the species of animal to which the recipientbelongs (i.e., “heterologous”), but the information may also be foreignonly to the particular individual recipient, or genetic informationalready possessed by the recipient. In the last case, the introducedgene may be differently expressed than is the native gene.

Example Mammalian SIRT1 Limits Replicative Lifespan in Response toChronic Genotoxic Stress

The Saccharomyces cerevisiae chromatin silencing factor Sir2 suppressesgenomic instability and extends replicative lifespan. In contrast, wefind that mouse embryonic fibroblasts (MEFs) deficient for SIRT1, amammalian Sir2 homolog, have dramatically increased resistance toreplicative senescence. Extended replicative lifespan of SIRT1-deficientMEFs correlates with enhanced proliferative capacity under conditions ofchronic, sub-lethal oxidative stress. In this context, SIRT1-deficientcells fail to normally up-regulate either the p19^(ARF)senescence-regulator or its downstream target p53. However, upon acuteDNA damage or oncogene expression, SIRT1-deficient cells show normalp19^(ARF) induction and cell-cycle arrest. Together, our findingsdemonstrate an unexpected SIRT1 function in promoting replicativesenescence in response to chronic cellular stress and implicatep19^(ARF) as a downstream effector in this pathway.

The Saccharomyces cerevisiae chromatin silencing factor Sir2 (SilentInformation Regulator 2) is an NAD-dependent histone deacetylase thatsuppresses transcription at several genomic loci (Blander and Guarente,2004). In addition, Sir2 suppresses recombination at the ribosomal DNAarray, thereby extending yeast lifespan (Blander and Guarente, 2004;Kaeberlein et al., 1999; Sinclair and Guarente, 1997). Because of itsNAD-dependence, the activity of Sir2 is linked to the energy status ofthe cell, providing a mechanism whereby cellular metabolism caninfluence lifespan. Over-expression or pharmacologic activation of Sir2homologs in Caenorhabditis elegans and Drosophila melanogaster alsoextends lifespan (Astrom et al., 2003; Rogina and Helfand, 2004;Tissenbaum and Guarente, 2001; Wood et al., 2004). Seven mammalian Sir2homologs, referred to as SIRT1-7, have been identified (Frye, 2000).Based on this conserved function in regulating lifespan in lowereukaryotes, mammalian Sir2 homologs have been proposed to play a similarrole (Kaeberlein et al., 1999). As SIRT1 is a nuclear protein and is themammalian homolog most highly related to Sir2, it has been the focus ofa large body of recent studies.

SIRT1-deficient mice suffer multiple abnormalities, including defects inspermatogenesis and in heart and retina development (Cheng et al., 2003;McBurney et al., 2003). The highly pleiotrophic phenotype ofSIRT1-deficient mice likely reflects the broad array of potential SIRT1substrates (reviewed in (Blander and Guarente, 2004)). In some cells,SIRT1 inhibits apoptosis in response to genotoxic stress and mayaccomplish this by several mechanisms. SIRT1 deacetylates the p53 tumorsuppressor protein (Cheng et al., 2003; Langley et al., 2002; Luo etal., 2001; Tissenbaum and Guarente, 2001; Vaziri et al., 2001), whichdown-regulates p53 via effects on stability and activity (Prives andManley, 2001). SIRT1 also inhibits apoptosis and promotes DNA repair bydeacetylating FOXO transcription factors (Brunet et al., 2004; Daitokuet al., 2004; Motta et al., 2004; Van Der Horst et al., 2004), and itinhibits Bax-induced apoptosis by deacetylating Ku70 (Cohen et al.,2004a). Expression of SIRT1 itself is activated by calorie restrictionand acute nutrient withdrawal, and thus, may promote cellular adaptationto metabolic stress (Cohen et al., 2004b; Nemoto et al., 2004). Asincreased stress resistance is a frequent correlate of longevity inmodel organisms (Finkel and Holbrook, 2000), the ability of SIRT1 tomodulate stress resistance in mammalian cells suggests a potential linkwith mammalian aging.

Cellular senescence has been employed as a model for mammalian aging(Campisi, 2000; Hayflick and Moorhead, 1961). This process, which can beinduced by several stimuli, consists of a state of permanent cell-cyclearrest associated with characteristic changes in cell morphology. Humanand mouse fibroblasts undergo a limited number of divisions in culture,eventually entering a state of cellular senescence known as replicativesenescence (Campisi, 2000; Hayflick and Moorhead, 1961). In humanfibroblasts, replicative senescence results from telomere attrition. Incontrast, mouse embryonic fibroblasts (MEFs), which possess much longertelomeres than human cells, undergo replicative senescence as a resultof sub-lethal oxidative damage incurred under standard cultureconditions (Busuttil et al., 2004; Parrinello et al., 2003; Sherr andDePinho, 2000; Wright and Shay, 2000). Cellular senescence also mayrepresent a tumor suppressor mechanism, preventing propagation of cellsthat incur potentially oncogenic mutations (Krtolica and Campisi, 2002).In this regard, a senescence-like state, termed premature senescence, isinduced by introduction of activated oncogenes into primary fibroblasts(Serrano et al., 1997). Likewise, acute DNA damage in primaryfibroblasts, as induced by various genotoxins, also triggers cellularsenescence (Chen et al., 1995; Robles and Adami, 1998; Sedelnikova etal., 2004).

In MEFs, the p53 protein plays a critical role in promoting senescencevia activation of a complex transcriptional program (Oren, 2003; Vousdenand Lu, 2002). Activation of p53 by the p19^(ARF) tumor suppressorprotein promotes replicative senescence. In this case, oxidative stresstriggers p19^(ARF) induction, which positively regulates p53 viainhibition of MDM2, a protein that mediates p53 degradation (Kurokawa etal., 1999; Pomerantz et al., 1998; Zhang et al., 1998; Zindy et al.,1998). Consistent with the importance of the p19^(ARF)/p53 pathway inreplicative senescence, spontaneous immortalization of MEFs duringculture usually results from adaptive mutations in p53 or silencing ofp19^(ARF) (Kamijo et al., 1997; Sherr and DePinho, 2000). Likereplicative senescence, premature senescence in response to activatedoncogenes also is mediated by p19^(ARF)-dependent activation of p53(Palmero et al., 1998). In this context, MEFs immortalized throughmutations in the p19^(ARF)/p53 pathway are transformed, rather thangrowth arrested, by introduction of activated oncogenes (Kamijo et al.,1997; Serrano et al., 1997). Finally, cellular senescence induced byacute DNA damage also is mediated by p53, but via a pathway that isindependent of p19^(ARF)(Kamijo et al., 1997; Stott et al., 1998).

Potential roles of SIRT1 with respect to replicative senescence inmammalian cells have not been elucidated. However, certain findings havesupported the notion that SIRT1, like yeast Sir2, might function toprevent senescence. In this regard, p53 is hyperacetylated inSIRT1-deficient MEFs (Cheng et al., 2003), and increased p53 acetylationhas been associated with senescence (Pearson et al., 2000). Likewise,over-expression of SIRT1 inhibits oncogene-induced premature senescencein MEFs (Langley et al., 2002). On the other hand, total p53 proteinlevels are reduced in SIRT1-deficient MEFs (Cheng et al., 2003), aneffect that could potentially inhibit senescence. Here, we have directlyassessed the effect of SIRT1-deficiency on replicative senescence ofMEFs. In marked contrast to Sir2 function in S. cerevisiae, we find thatSIRT1 promotes replicative senescence in MEFs and that p19^(ARF) is anovel downstream effector of SIRT1 in this pathway.

Results

SIRT1-Deficiency Abrogates Replicative Senescence.

We have previously described SIRT1-deficient (referred to as S1KO) miceand S1KO MEFs (Cheng et al., 2003). To explore the role of SIRT1 inreplicative senescence, we subjected S1KO and WT control MEFs to serialpassage according to a 3T3 protocol (Todaro and Green, 1963). Aspreviously shown, WT cultures underwent growth arrest after 5-8 passages(FIG. 1A) (Todaro and Green, 1963). Surprisingly, rather than arrestingearlier than WT cells, S1KO cells did not undergo replicative senescenceand continued to proliferate unabated (FIG. 1A). Similar results wereobtained with 6 independent S1KO MEF lines (FIG. 1A, data not shown).The replicative capacity of MEFs can also be assessed by their abilityto form colonies when seeded at low density. In this assay, WT MEFcultures underwent senescence before forming visible colonies, aspreviously described (Bardeesy et al., 2002; Sage et al., 2000), whereasS1KO MEF lines exhibited significantly greater clonogenic potential(FIG. 1B). Similar results were obtained for 4 independent S1KO and 3independent WT MEF lines. Together, these observations suggest thatSIRT1-deficiency extends, rather than shortens, the normal replicativelifespan of primary mouse fibroblasts.

The increased replicative lifespan of SIRT1-deficient MEFs might be dueto either an acute effect of SIRT1-deficiency or to secondaryadaptations during growth of the S1KO cells in utero or in culture. Todistinguish these possibilities, we acutely inactivated SIRT1 incultured MEFs. We previously described mice in which exon 4 of SIRT1 wasflanked by LoxP sites (ex4^(Flox)) and showed that mice harboringhomozygous deletions of this exon were phenotypically indistinguishablefrom S1KO mice (Cheng et al., 2003). This finding indicates that thetruncated SIRT1 protein generated by the exon 4 deletion allele isnon-functional. Ex4^(Flox)/S1KO and ex4^(Flox)/WT MEF lines weregenerated from crosses of ex₄ ^(Flox)/ex₄ ^(Flox) mice and miceheterozygous for the SIRT1 null allele (WT/S1KO). Adenoviraltransduction of these MEFs with Cre Recombinase fused to GreenFluorescent Protein (GFP-Cre) resulted in nearly complete excision offloxed exon 4 and replacement of the full-length SIRT1 protein by thenon-functional, faster-migrating Δex4 protein (FIG. 1C). 3T3 serialpassage of multiple lines, following treatment with GFP-Cre or GFPcontrol, demonstrated that the GFP-Cre-treated Δex4^(Flox)/S1KO cultureswere strikingly resistant to replicative senescence, as compared to thesame lines treated with GFP control virus or Δex4^(Flox)/WT MEFs treatedwith either GFP-Cre or GFP control virus (FIG. 1E). Similar results wereobtained with 6 additional ex4^(Flox)/S1KO MEF lines and 3 additionalex4^(Flox)/WT MEFs lines, in either serial passage or colony formationexperiments (FIG. 6). Thus, acute inactivation of SIRT1 in MEFs confersresistance to replicative senescence, demonstrating that this is adirect effect of loss of SIRT1 function.

To further exclude potential secondary mutations as causes of theenhanced replicative lifespan of S1KO MEFs, we asked whether the effectof SIRT1-deficiency on replicative lifespan could be reversed byexogenous SIRT1 expression. Recombinant SIRT1 was introduced into S1KOMEFs by retroviral transduction at passage 7, a point by which WTcultures had already senesced. Transduced SIRT1 expression occurred atlevels slightly lower than those of endogenous SIRT1 (FIG. 1E). UponSIRT1 reconstitution, the S1KO MEF cultures showed significantly reducedproliferative capacity compared to control cultures treated with emptyvirus (FIG. 1F, and data not shown). Similar results were obtained withmultiple independent late-passage S1KO MEF cultures in either serialpassage or colony formation assays (FIG. 6). Notably, the deacetylaseactivity of SIRT1 is required for reversal of the enhanced proliferationof S1KO MEFs, since a catalytically inactive SIRT1 mutant protein failedto rescue the S1KO phenotype (FIG. 6E). Overall, these findings providestrong evidence that the resistance of S1KO MEFs to replicativesenescence is a direct consequence of SIRT1-deficiency, and not due tosecondary immortalizing mutations. This conclusion is further supportedby our finding that SIRT1 reconstitution also led to up-regulation of adownstream regulator of replicative senescence (see below).

p19^(ARF) is a Novel Downstream Effector of SIRT1 in Regulation ofReplicative Senescence

We previously showed that S1KO MEFs exhibited normal sensitivity toionizing radiation, adriamycin, and to UV (Cheng et al., 2003) (FIG. 7).Thus, even though S1KO MEFs are resistant to replicative senescence,they retain normal cell-cycle control in response to various forms ofDNA damage. Because p19^(ARF) is a critical senescence regulator, butdoes not function in acute DNA damage responses (Kamijo et al., 1997;Stott et al., 1998), we asked whether SIRT1 regulates p19^(ARF) levels.Western analysis of p19^(ARF) levels in early passage (P3) S1KO and WTMEFs revealed that base-line levels of the p19^(ARF) protein weresignificantly lower in S1KO cells compared to WT cells (FIG. 2A, lanes 1and 3). In contrast, there was no difference in p19^(ARF) levels in WTand S1KO cells following treatment with the DNA damage-inducing agentadriamycin (FIG. 2A, lanes 2 and 4). Over the course of serial passage,p19^(ARF) accumulates in WT MEFs as previously described (Zindy et al.,1998); however, in S1KO MEFs, p19^(ARF) accumulation was significantlyattenuated (FIG. 2B). The p19^(ARF) protein regulates senescence, atleast in part, by stabilizing p53 (Kurokawa et al., 1999; Pomerantz etal., 1998). Consistent with this function of p19^(ARF), S1KO cells alsoshowed attenuated accumulation of p53 over serial passage (FIG. 2B). Onthe other hand, p53 was hyperacetylated in S1KO cells both following DNAdamage (FIG. 2A; (Cheng et al., 2003) and also during serial passage(FIG. 2B). Thus, SIRT1-deficiency has two distinct effects on p53:hyperacetylation and reduction in total levels.

Because silencing of p19^(ARF) expression is commonly observed duringthe spontaneous immortalization of WT MEFs that occurs upon extendedpassage in culture (Kamijo et al., 1997), reduced p19^(ARF) levels inS1KO MEFs theoretically might reflect such a random event rather than adirect functional consequence of absence of SIRT1 expression. To addressthis issue, we asked whether reconstitution of late passage S1KO cellswith SIRT1 expression could reverse the reduced p19^(ARF) levels, inaddition to reversing their enhanced replicative potential (see FIG.1F). In three independent experiments, reconstitution of S1KO MEFs atpassage 7 resulted in increased levels of p19^(ARF) (FIG. 2C) and p53(data not shown). Therefore, we conclude that the reduced p19^(ARF)levels in S1KO MEFs results from loss of a novel SIRT1 function, ratherthan from secondary adaptive mutations or silencing events.

Although p19^(ARF) regulates p53 stability, p53 also appears to regulatep19^(ARF) in a negative feedback loop (Quelle et al., 1995). BecauseSIRT1-deficiency leads to p53 hyperacetylation, the resulting potentialincrease in p53 activity might, in theory, lead to selection for cellsthat have down-regulated p19^(ARF). To explore this possibility, we bredmice with homozygous inactivating mutations in both SIRT1 and p53 andthen generated S1KO/p53-deficient MEFs. Assays of the doubly-deficientMEFs demonstrated that, even in a p53-deficient background,SIRT1-deficiency led to reduced p 9^(ARF) protein levels (FIG. 2D).Thus, the reduced levels of p19^(ARF) in S1KO cells cannot be attributedto feedback from p53 hyperacetylation or an adaptation to putative p53hyperactivity. Together, our observations suggest that SIRT1 has a novelfunction in regulating replicative senescence by promoting up-regulationof p19^(ARF).

SIRT1-Deficient Cells Show Enhanced Proliferative Capacity underConditions of Chronic, Sub-Lethal Oxidative Stress

Because replicative senescence in MEFs is thought to be a response tothe non-physiologic levels of oxidative stress present in standard cellculture conditions (Parrinello et al., 2003), we asked whether and inwhat context SIRT1 regulates oxidative stress resistance. S1KO and WTcultures were treated with a wide range of doses of H₂O₂ for 15 minutes,transferred to normal media for 18 hours, and the percentage of S-phasecells assessed by BrdU incorporation to determine the ratio of BrdUincorporation in H₂O₂-treated cells compared to control treated cells(S-phase ratio). At all doses assayed, the S-phase ratios of S1KO cellsand WT control cells were indistinguishable (FIG. 3A). These dataindicated that, under the conditions of this assay, SIRT1-deficiencydoes not confer resistance to acute oxidative stress. This observationwas somewhat unexpected, given that replicative senescence is in fact aresponse to oxidative stress. However, since replicative senescencereflects a cellular response to continuous culture under mild chronicoxidative stress, it is conceivable that such conditions might triggerdifferent cellular responses than acute oxidative insults.

To test whether S1KO MEFs have enhanced replicative potential underconditions of chronic, sub-lethal oxidative stress, we tested them forgrowth in the continuous presence of 50 uM H₂O₂ or under serumdeprivation, conditions which led to mild oxidative stress andaccelerated cell-cycle arrest/senescence of WT MEFs (Nemoto and Finkel,2002); (FIG. 8A). Under both these conditions, S1KO MEFs proliferatedsignificantly better than WT MEFs (FIG. 3B), and accumulated lowerlevels of p19^(ARF) (FIG. 3E). Further, at the end of 1 week of cultureunder these conditions, greater than 50% of WT MEFs stained positive forthe senescence marker SA-β-galactosidase; whereas only about 10% of S1KOMEFs were SA-β-galactosidase positive (FIG. 3C). In these experiments,S1KO or WT MEFs did not undergo apoptosis as assessed by annexin Vstaining (data not shown). Together, these observations indicate thatthe resistance of S1KO MEFs to replicative senescence reflects a novelfunction of SIRT1 in controlling proliferative capacity under conditionsof chronic, sub-lethal oxidative stress.

SIRT1-Deficient Cells Retain Intact Responses to Oncogenic Ras

We next asked whether SIRT1 regulates p19^(ARF) levels and prematuresenescence of MEFs in response to oncogenic Ras. The activated Rasmutant H-Ras^(V12) (or empty virus control) was introduced into S1KO andWT MEFs by retroviral transduction, and cell-cycle arrest assessed byBrdU incorporation. Unlike immortal 3T3 MEF lines (Kamijo et al., 1997),S1KO MEFs showed entirely normal responses to H-Ras^(V12) with respectto cell-cycle arrest (FIG. 4A). We also carried out soft agar assays foranchorage-independent growth, a characteristic of cellulartransformation. When transduced with H-Ras^(V12), S1KO MEFs failed toform colonies in soft agar, like primary WT MEFs; whereasH-Ras^(V12)-expressing immortal 3T3 lines exhibited robustanchorage-independent colony formation (FIG. 4B). Moreover, there was nosignificant difference in levels of p19^(ARF) induction in S1KO and WTMEFs (FIG. 4C), indicating that SIRT1 is dispensable for p19^(ARF)induction in response to H-Ras^(V12)-expression. Similar results wereobtained for three independent S1KO MEF lines. Thus, SIRT1 is requiredfor normal p19^(ARF) induction and cellular senescence in response toprolonged replication but is dispensable for senescence in MEFsfollowing acute expression of oncogenic Ras.

Notably, S1KO cells were sensitive to H-Ras^(V12)-induced senescence andresistant to H-Ras^(V12)-induced transformation, even at very latepassages (passage 50) (FIG. 4B). This is in sharp contrast to the cellsthat grow out of WT MEF cultures following such extended passage, whichare transformed by activated H-Ras^(V12) (FIG. 4B). In this context,cells that have grown out of WT MEF cultures by passage 50 had, asexpected (Kamijo et al., 1997), either lost p53-dependent induction ofthe cell-cycle regulator p21 (FIG. 4C, WT2-3T3) or expression ofp19^(ARF)(FIG. 4C, WT1-3T3) in response to H-Ras^(V12). In contrast,S1KO cultures at passage 50 (FIG. 4C, KO-3T3) showed normal p19^(ARF)and p21 induction in response to H-Ras^(V12). S1KO MEFs also arrestednormally in response to acute expression of p19^(ARF) (FIGS. 9A and B),indicating that the pathway downstream of p19^(ARF) is functionallyintact. We also found that the p16^(INK4A)/pRb pathway was intact inSIRT1KO MEFs (FIG. 9B). In addition, S1KO MEFs also were not transformedby oncogenic c-myc, in contrast to immortal MEF lines carrying adaptivemutations (FIGS. 9C and D). We conclude that SIRT1-deficiency allows forcontinued proliferation of MEFs without subjecting them to selection forimmortalizing mutations.

SIRT1 Regulation of Cellular Lifespan via p19^(ARF)

Our findings lead to the surprising conclusion that mammalian SIRT1 andbudding yeast Sir2 have opposite effects on replicative senescence.Thus, while Sir2 extends replicative lifespan in budding yeast, SIRT1functions to limit the replicative lifespan of MEFs. We also note that,similar to our results with MEFs, RNAi-mediated knock-down of SIRT1dramatically extends replicative lifespan of human primary fibroblasts(E. Michishita, I. Horikawa, and J. C. Barret, personal communication),documenting the generality of this function across mammalian species. Wealso show that alleviation of replicative senescence in the absence ofSIRT1 correlates with up-regulation of p19^(ARF). Because p19^(ARF)promotes stabilization of p53, SIRT1-deficiency in MEFs also impairsnormal up-regulation of p53 levels during prolonged culture, whichlikely accounts for bypass of senescence. However, in contrast to manymutations that confer resistance to replicative senescence,SIRT1-deficiency in MEFs leaves intact cell-cycle arrest/senescenceresponses to acute DNA damage, acute oxidative stress, and activatedoncogenes. Overall, our findings also support the notion that mammaliancells have evolved different mechanisms to respond to chronic versusacute genotoxic insults (FIG. 5).

Several lines of evidence indicate that the effects of SIRT1-deficiencyon p19^(ARF) and replicative senescence are not due to adaptive changesin S1KO cultures. First, acute inactivation of SIRT1 by Cre-Loxstrategies confers resistance to senescence. Second, the enhancedreplicative potential and attenuated p19^(ARF) levels of S1KO cells canbe reversed by exogenous SIRT1, even at relatively late passage, whenwild-type cultures have already senesced. Third, SIRT1-deficiency leadsto reduced p19^(ARF) levels in a p53-deficient background, in whichthere should be no selection pressure for cells expressing low levels ofp19^(ARF). Fourth, unlike immortalized cell lines that have acquiredsecondary adaptations in culture, SIRT1-deficient MEFs retain fullyfunctional responses to activated oncogenes and acute DNA damage thatare indistinguishable from those of primary wild-type MEFs. We have alsofound that although SIRT1-deficient MEFs bypass senescence, they do notaccumulate genomic instability beyond that observed in wild-type cells(FIG. 10). These results are consistent with data found in p19^(ARF)deficient MEFs, where cellular immortalization is not accompanied byincreased genomic instability (Kamijo et al., 1997; Zindy et al., 1997).Thus, the similarity of the phenotypes resulting from p19^(ARF)- andSIRT1-deficiency with respect to extending MEF lifespan in the absenceof increased genomic instability further supports our conclusion thatthe effect of SIRT1-deficiency on MEF replicative lifespan ultimately ismediated through p19^(ARF). Together, these findings argue that theresistance of S1KO cells to replicative senescence reflects a novelfunction of SIRT1 that influences regulation of p19^(ARF) expression inresponse to chronic genotoxic stress.

Differential Regulation of p19^(ARF) by SIRT1 Distinguishes Replicativefrom Oncogene-Induced Senescence

The p19^(ARF) protein is a critical regulator of cellular senescence;correspondingly, it is up-regulated during both replicative senescenceand oncogene-induced premature senescence (Palmero and Serrano, 2001;Zindy et al., 1998). It has been proposed that replicative senescenceand oncogene-induced senescence may not be functionally distinct(Ben-Porath and Weinberg, 2004). In this context, both processes arethought to result from increased cellular levels of reactive oxygenspecies (ROS) (Irani et al., 1997; Lee et al., 1999; Parrinello et al.,2003; Sundaresan et al., 1996). However, we now show that SIRT1 isrequired for normal up-regulation of p19^(ARF) during replicativesenescence in response to chronic, sub-lethal genotoxic stress; but itis not required for activated Ras-induced premature senescence. Oneconceivable mechanism by which these two processes might differ would beby triggering different levels of ROS. However, our preliminary datasuggests we found that both forms of stress induction in MEFs led tosimilar intracellular levels of ROS (FIG. 8). Thus, the differentialinvolvement of SIRT1 in replicative versus oncogene-induced senescencepoints to a fundamental distinction in the pathways underlying thesephenomena, even though both are ultimately mediated by p19^(ARF) (Seemodel, FIG. 5). In this regard, while genotoxic stress is the primarytrigger for replicative senescence, increased ROS levels is only one ofseveral consequences of mitogenic signaling evoked by activated Ras(Irani et al., 1997).

We can tentatively rule out several potential mechanisms by which SIRT1might regulate p19^(ARF) in the context of replicative senescence. Inparticular, SIRT1 does not function directly to deacetylate p19^(ARF)based on assays with available anti-acetyl lysine antibodies (data notshown). Also, direct regulation of histones at the p19^(ARF) promoter bySIRT1 seems unlikely to account for reduced p19^(ARF) levels in S1KOcells; since histone hyperacetylation should increase, rather thandecrease, p19^(ARF) expression. Thus, SIRT1 may target, directly orindirectly, an upstream p19^(ARF) regulator. Such a putative SIRT1substrate could be part of the senescence response to chronic genotoxicstress, or alternatively, could regulate the of induction the senescenceresponse itself. In the latter context, the lack of p19^(ARF)accumulation in S1KO cells could simply reflect the failure of thesecells to accumulate a threshold level of genotoxic stress required toinitiate the replicative senescence response.

The regulation of p19^(ARF) by SIRT1 could occur transcriptionally orpost-transcriptionally. Several negative regulators of replicativesenescence that regulate p19^(ARF) transcription have been identifiedincluding the polycomb group proteins Bmi-1, Cited2, Twist, thetranscription factors Tbx2 and Tbx3 (Carlson et al., 2002; Jacobs etal., 2000; Jacobs et al., 1999; Krane et al., 2003), and the recentlyidentified Pokemon factor (Maeda et al., 2005). However, unlike theputative SIRT1-regulated factor, negative regulation of p19^(ARF) bythese factors counteracts Ras-induced premature senescence andcooperates with Ras to promote oncogenic transformation. In addition,our preliminary experiments have not revealed hyperacetylation ofseveral of these known factors in S1KO cells. At thepost-transcriptional level, there is evidence that p19^(ARF) proteinundergoes ubiquitin-dependent degradation (Kuo et al., 2004), and SIRT1might deacetylate a factor involved in regulating p19^(ARF) stability.However, the E2/E3 ubiquitination factors that regulate p19^(ARF)turnover have yet to be identified. Thus, the identity of the putativeSIRT1-target that influences replicative senescence remains unknown.

Our findings demonstrate that SIRT1 expression can influence p53function via two distinct mechanisms, which have opposite effects on netp53 activity. Thus, SIRT1 inactivates p53 by deacetylation, but can leadto p53 induction via regulation of p19^(ARF). In the context ofreplicative senescence in S1KO MEFs, reduced p53 levels outweigh theeffects of p53 hyperacetylation. In contrast, following acute DNA damagein SIRT1-deficient thymocytes, p53 levels are unaffected, and p53hyperacetylation renders the cells hypersensitive to DNA damage (Chenget al., 2003). Similarly, over-expression of SIRT1 deacetylates p53 inMEFs following activated Ras expression and, thereby, appears toattenuate the senescence response (Langley et al., 2002). Therefore, aswe observed no effect of SIRT1-deficiency on total p19^(ARF) and p53levels following activated Ras expression in MEFs (FIG. 4C, and data notshown), p53 hyperacetylation in these cells might be predicted to leadto a hyperactive senescence response. However, we could not assay forsuch a hyperactive response, because premature senescence of wild-typeMEFs occurs almost immediately following transduction and selection foractivated Ras-expression. Overall, our current findings, coupled withprevious work, suggest that the effect of SIRT1 on net cellular p53activity may be dependent on both cell type and the context of cellularstress.

Perspective: Cellular Senescence, Mammalian Aging and SIRT1.

Based on the ability of Sir2 to extend lifespan in lower eukaryotes,SIRT1 has been considered an attractive pharmacological target inhumans. However, the divergence of yeast Sir2 and mammalian SIRT1functions in replicative senescence highlights potential difficultieswith this generalization. In this regard, the relationship betweencellular senescence and organismal aging clearly differs in unicellularand multi-cellular organisms. In unicellular yeast, cellular senescenceis, by definition, a detrimental process, while in mammals cellularsenescence shows antagonistic pleiotropy; that is, it may be beneficialin some contexts, such as tumor suppression, but detrimental in otherssuch as promoting aging of mitotic tissues (Campisi, 1997). Moreover, asour findings indicate that SIRT1 has different effects on senescencedepending on the particular trigger, a detailed examination of itsfunction in specific contexts would be critical in attempts to predicthow modulation of SIRT1 activity would impact mammalian physiology andaging.

Regardless of the role of SIRT1 in normal physiology, our finding that“immortal” SIRT1-deficient MEFs have intact responses to acute genotoxicstress and oncogenic transformation indicates that modulation of SIRT1activity might be useful to grow large quantities of certain cell typesfor experimental or therapeutic purposes without selecting fortransforming mutations. Indeed, inactivation of p19^(ARF) can augmentexpansion and long-term cultivation of mouse hepatocytes, and contributeto liver regeneration following transplantation (Mikula et al., 2004).In addition, inhibition of p19^(ARF) expression can promote theself-renewal of hematopoietic stem cells, augmenting ex vivo expansionand in vivo repopulating capacity (Iwama et al., 2004). Thus, in these,and potentially other cell types, SIRT1 might be a useful target forpharmacologic modulation.

Materials and Methods

Culturing and Retroviral Reconstitution of MEFs and 3T3 SerialPassaging.

MEFs were cultured in DMEM supplemented with 15% fetal calf serum, 8 mMnonessential amino acids, 8 mM sodium pyruvate, 9 mM glutamine, 9 mMpenicillin/streptomycin, 18 mM HEPES (pH7), and 0.006 mMbeta-mercaptoethanol. For replicative senescence assays, a 3T3 protocol(Sherr and DePinho, 2000; Todaro and Green, 1963) was followed bycounting cells in triplicate and re-seeding 3×10⁵ cells per 10 cm plateevery 3 days. For colony formation assays. MEFs were seeded at a densityof 10³ cells per 10 cm plate, cultured for two weeks, and coloniesstained with crystal violet. Retroviral packaging and infection wasperformed as previously described (Cheng et al., 2003). For chronic H₂O₂cultures, 1×10⁶ cells were plates in 2 ml media with 50 uM H₂O₂ lackingbeta-mercaptoethanol in 6 well plates, and media was replaced everyday.

Generation and Cre-Deletion of Conditionally Targeted SIRT1 MEFs.

Gene-targeting to generate ex4^(Flox) ES cells was previously described(Cheng et al., 2003). ES cell clones were injected into C57BL/6blastocysts, founder chimeras bred to 129/Sv females, and F1heterozygotes intercrossed. Resulting ex4^(Flox)/ex4^(Flox) mice werecrossed to WT/S1KO mice, and MEFs were isolated from embryonic day 13.5embryos and genotyped by Southern analysis. For Cre deletion,conditionally targeted SIRT1 MEFs were infected with Cre-GFP and GFPadenoviruses (gift of Jonathan Walsh and Richard Mulligan). Briefly,1×10⁶ cells were plated with 1×10⁸ viral particles (MOI=100). After 24hrs, cells were washed, and fresh medium was added. At 48 hrpost-infection, the efficiency of infection was calculated based onpercentage of GFP positive cells. 48 hr. later, cells were count andplate for the 3T3 passage and colony formation assays.

Western Analysis.

Western analysis was carried out as previously described (Cheng et al.,2003). Antibodies: anti-p19 (Abcam, ab80); anti-p16 (Santa Cruz);anti-SIRT1 (Upstate); anti-□-tubulin (Sigma); anti-p53 (CM-5,Novocastra); anti-p21 (Ab-4, Oncogene, Ab-4). p53 acetylation wasassessed using PAbLys(Ac)379m antibody, as previously described (Chenget al., 2003).

Soft Agar Assays.

Anchorage-independent growth was assessed by soft agar assays, aspreviously described (Dannenberg et al., 2000). Briefly, 2.5×10⁴ cellswere resuspended in 2 ml 0.4% low melting point agarose (Sigma) in DMEMwith 13% serum, and seeded into 6-well plates coated with 1% low meltingpoint agarose in DMEM with 10% serum. Foci were scored and photographedafter 14 days.

Cell Cycle Analysis.

BrdU incorporation was assayed with anti-BrdU antibodies (BD Pharmingen)according to the manufacturer's instructions. Briefly, 5×10⁵ cells werepulsed with BrdU for 4 hours, harvested, stained with FITC-conjugatedanti-BrdU antibodies and propidium iodide, and cell-cycle profilesanalyzed by flow cytometry.

REFERENCES

All references cited herein are hereby incorporated by reference.

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1. A method of overcoming replicative senescence of mammalian cells,comprising culturing the cells in the presence of an active agent orcompound that inhibits the expression of SIRT1, wherein the agent orcompound is a RNA interfering agent and/or a peptide nucleic acid (PNA);and wherein said cultured cells are resistant to replicative senescence.2. The method of claim 1, wherein the RNA interfering agent is a doublestranded, short interfering RNA (siRNA).
 3. The method of claim 2,wherein the siRNA is about 15 to about 28 nucleotides in length.
 4. Themethod of claim 2, wherein the siRNA is about 19 to about 25 nucleotidesin length.
 5. The method of claim 2, wherein the siRNA is about 21nucleotides in length.
 6. The method of claim 2, wherein said siRNA isdouble-stranded and comprises a 3′ overhand on each strand.
 7. Themethod of claim 2, wherein said siRNA inhibits SIRT1 by transcriptionalsilencing.
 8. The method of claim 1, wherein the cells are stem cells.9. The method of claim 8, wherein the cells are somatic stem cells. 10.The method of claim 9, wherein the somatic stem cells are neuronal stemcells.
 11. The method of claim 1, wherein the cells are human cells. 12.The method of claim 1, wherein the cells are murine cells.
 13. Themethod of claim 1, wherein the cells in which SIRT1 is inhibited arefurther cultured to undergo at least one mitotic cell division.
 14. Themethod of claim 1, wherein the cells in which SIRT1 is inhibited arefurther cultured to undergo at least ten mitotic cell divisions.
 15. Themethod of claim 1, wherein the cultured cells are capable ofself-renewal and expansion in culture, and have the potential todifferentiate into cells of other phenotypes.